Download Novex® Pre-Cast Gel Electrophoresis Guide

Novex® Pre-Cast Gel
Electrophoresis Guide
General information and protocols for using
Novex® pre-cast gels
Rev. date: October 2010
Manual part no. IM-1002
MAN0003187
User Manual
Contents
General Information.................................................................................................................................... v
Overview of Electrophoresis.......................................................................................................................1
Novex® Pre-Cast Gels ................................................................................................... 4
Novex® Gel Specifications ...........................................................................................................................4
Gel Selection..................................................................................................................................................6
Well Volume .................................................................................................................................................8
Gel Staining ...................................................................................................................................................9
Methods ....................................................................................................................... 10
General Guidelines for Preparing Samples and Buffers.......................................................................10
Tris-Glycine Gels ........................................................................................................................................12
Tricine Gels..................................................................................................................................................14
Zymogram Gels ..........................................................................................................................................16
IEF Gels........................................................................................................................................................19
ZOOM® Gels ...............................................................................................................................................24
TBE Gels.......................................................................................................................................................26
TBE-Urea Gels.............................................................................................................................................28
DNA Retardation Gels...............................................................................................................................30
Electrophoresis of Novex® Pre-Cast Gels................................................................................................32
Power Supply Settings for Novex® Pre-Cast Gels .................................................................................33
Opening Novex® Pre-Cast Gel Cassettes ................................................................................................34
Coomassie Staining ....................................................................................................................................35
Silver Staining .............................................................................................................................................39
SYPRO® Ruby Staining ..............................................................................................................................44
SYBR® Green Staining................................................................................................................................47
Ethidium Bromide Staining ......................................................................................................................48
Gel Drying ...................................................................................................................................................49
Blotting Novex® Pre-Cast Gels .................................................................................................................52
Calibrating Protein Molecular Weight ....................................................................................................58
Troubleshooting..........................................................................................................................................61
iii
Appendix...................................................................................................................... 63
Accessory Products ....................................................................................................................................63
Recipes .........................................................................................................................................................65
Gel Migration Charts .................................................................................................................................72
Technical Support.......................................................................................................................................76
References....................................................................................................................................................78
iv
General Information
Purpose of the
Guide
A variety of pre-cast gels for use with the XCell SureLock™ Mini-Cell are available
from Invitrogen. These include gels for analysis of proteins (Tris-Glycine,
Tricine, Zymogram, IEF, and ZOOM® Gels) and nucleic acids (TBE, TBE-Urea,
and DNA Retardation).
The Novex® Pre-Cast Gel Electrophoresis Guide contains information about the
Novex® Pre-Cast gels and is intended to supplement the Gel Instruction Cards
(IM-6000 to IM-6008) supplied with the pre-cast gels. Complete protocols for
sample and buffer preparation, electrophoresis conditions, staining, and blotting
are provided in this guide.
To request the instruction cards or for additional information, call Technical
Support (see page 76) or download the manuals from our website at
www.invitrogen.com.
Storage and Shelf
life
Store Novex® Pre-Cast Gels at +4°C. The gels have a shelf life of 4–8 weeks
depending upon the gel type when stored at +4°C.
Do not freeze Novex® Pre-Cast Gels.
Use gels immediately from the refrigerator. Extended exposure of the gels to
room temperature significantly impairs the performance of the gel.
Packaging
The Novex® Pre-Cast Gels are supplied as 10 gels per box. Gels are individually
packaged in clear pouches with 4–10 mL of Packaging Buffer.
Handling the Gels
The Packaging Buffer contains 0.02% sodium azide and residual acylamide
monomer. Wear gloves at all times when handling gels.
Warning: This product contains a chemical (acrylamide) known to the state of
California to cause cancer. Refer to the Invitrogen website for the SDS (see page
76).
Intended Use
For research use only. Not intended for human or animal diagnostic or
therapeutic uses.
v
Overview of Electrophoresis
Introduction
Electrophoresis is a simple, rapid, and sensitive analytical tool for separating
proteins and nucleic acids based on their physical characteristics (mass,
isoelectric point, etc.).
Most biological molecules carry a net charge at any pH other than their
isoelectric point and migrate at a rate proportional to their charge density in an
electrical field.
The mobility of a biological molecule through an electric field depends on the
following factors:

Field strength

Net charge on the molecule

Size and shape of the molecule

Ionic strength

Properties of the medium through which the molecules migrate (e.g.,
viscosity, pore size)
Support Matrix
Polyacrylamide and agarose are two types of support matrices used in
electrophoresis. The support matrix is a porous media that acts as a molecular
sieve. The sieving function depends on the pore size, and concentration of the
matrix. Agarose has a large pore size and is ideal for separating macromolecules such as nucleic acids and protein complexes. Polyacrylamide has a
smaller pore size and is ideal for separating proteins and smaller nucleic acids.
Polyacrylamide Gel
Electrophoresis
(PAGE)
Polyacrylamide gels are formed by the polymerization of acrylamide
monomers into long chains, crosslinked by bifunctional compounds such as
N,N-methylene-bisacrylamide (bis) that react with the free functional groups at
the chain termini.
The pore size of the gel is governed by the concentration of acrylamide and
bisacrylamide (%T and %C).
%T = concentration of total monomer
%C = proportion of cross linker (as a percentage of total monomer)
The higher the acrylamide concentration, the smaller the pore size, allowing
resolution of low molecular weight molecules and vice-versa.
Continued on next page
1
Overview of Electrophoresis, Continued
Buffer Systems
Electrophoresis is performed using continuous or discontinuous buffer systems.
Continuous buffer systems utilize a single buffer for the gel and the running
buffer.
Discontinuous buffer systems (Ornstein 1964) utilize different gel buffers and
running buffer. In addition, two gel layers of different pore size, the stacking
and separating gel, are used. Electrophoresis using a discontinuous buffer
system allows concentration of the sample to a narrow region prior to
separation, resulting in sharper bands and higher resolution.
Electrophoresis
Sample Conditions
Depending upon the application, electrophoresis can be performed under the
following conditions:
Denaturing
Electrophoresis is performed under denaturing conditions using an anionic
detergent such as sodium dodecylsulfate (SDS). SDS denatures and unfolds the
proteins by binding the hydrophobic portions of the protein at a ratio of ~1.4 g
SDS per gram of protein. The resultant SDS-protein complexes are highly
negatively charged and migrate through the gel based on their size rather than
charge.
Non-Denaturing (Native)
Electrophoresis is performed under non-denaturing (native) conditions using
buffer systems that maintain the native protein conformation, cohesion of
subunits, and biological activity. During native electrophoresis, proteins are
separated based on their charge to mass ratios.
Reducing
Electrophoresis is performed under reducing conditions using reducing agents
such as dithiothreitol (DTT) or -mercaptoethanol (-ME). The reducing agents
cleave any disulfide bonds between cysteine residues resulting in complete
separation of denatured proteins into their individual subunits.
Continued on next page
2
Overview of Electrophoresis, Continued
Power Supply
Considerations for
Electrophoresis
In electrical terms, the process of electrophoresis is closely associated with the
following equations derived from Ohm’s Law:
Voltage = Current × Resistance (V=IR)
Wattage = Current × Voltage (W=IV)
Resistance
The electrical resistance of the assembled electrophoresis cell is dependent on
buffer conductivity, gel thickness, temperature, and the number of gels being
run. Although the resistance is determined by the gel system, the resistance
varies over the course of the run.

In discontinuous buffer systems (and to a lesser extent in continuous buffer
systems) resistance increases over the course of electrophoresis. This occurs
in the Tris-Glycine buffer system as highly conductive chloride ions in the
gel are replaced by less conductive glycine ions from the running buffer.

Resistance decreases as the temperature increases.
Voltage
The velocity of an ion in an electric field varies in proportion to the field
strength (Volts per unit distance). The higher the voltage, the faster an ion
moves. For most applications, we recommend a constant voltage setting.

A constant voltage setting allows the current and power to decrease over
the course of electrophoresis, providing a safety margin in case of a break
in the system.

The constant voltage setting does not need adjustment to account for
differences in number or thickness of gels being electrophoresed.
Current
For a given gel/buffer system, at a given temperature, current varies in
proportion to the field strength (voltage) and/or cross-sectional area (thickness
and/or number of gels). When using a constant current setting, migration starts
slow, and accelerates over time, thus favoring stacking in discontinuous gels.
When running under constant current, set a voltage limit on the power supply
at, or slightly above the maximum expected voltage to avoid unsafe conditions.
At constant current voltage increases as resistance increases. If a local fault
condition occurs (e.g., a bad connection), high local resistance may cause the
voltage to reach the maximum for the power supply, leading to overheating
and damage of the electrophoresis cell.
Power
Wattage measures the rate of energy conversion, which is manifest as heat
generated by the system. Using constant power ensures that the total amount of
heat generated by the system remains constant throughout the run, but results
in variable mobility since voltage increases and current decreases over the
course of the run. Constant power is typically used when using IEF strips.
When using constant power, set the voltage limit slightly above the maximum
expected for the run. High local resistance can cause a large amount of heat to
be generated over a small distance, damaging the electrophoresis cell and gels.
3
Novex® Pre-Cast Gels
Novex® Gel Specifications
Introduction
The Novex® Pre-Cast Gel cassette is 10 cm × 10 cm in size, and designed for use
with the XCell SureLock™ Mini-Cell and XCell6™ MultiGel Unit (see page 63 for
ordering information).
Novex® Pre-Cast Gels are available for resolving proteins in the range of
2–500 kDa and nucleic acids in the range of 10–3,000 bp, depending upon the
type and acrylamide percentage of the gel. Refer to Gel Selection (page 6) for
details on applications and migration patterns.
Specifications
Gel Matrix:
Acrylamide/Bisacrylamide
Gel Thickness:
1.0 mm or 1.5 mm
Gel Size:
8 cm × 8 cm
Cassette Size:
10 cm × 10 cm
Cassette Material:
Styrene Copolymer (recycle code 7)
Sample Well Configuration:
1, 5, 9, 10, 12, 15-well, 2D-well, and IPG well
Continued on next page
4
Novex® Gel Specifications, Continued
Novex® Gel
Formulations
Gel Type
All Novex® Pre-Cast gels are made with high purity reagents. The gels for DNA
analysis are DNase-free. The composition of the different gels is listed below:
Formulation
Stacking Gel
Separating Gel
% BisAcrylamide
pH
Tris-Glycine Gels
(except 4%)
Tris-base, HCl,
Acrylamide, Bisacrylamide, TEMED,
APS, Ultrapure water
4%
6%, 8%, 10%, 12%,
14%, 16%, 18%,
4–12%, 8–16%,
4–20%, 10–20%
2.6%
8.6
4% Tris-Glycine Gels
Same as Tris Glycine
3.5%
4%
1.3%
8.6
Tricine Gels
Tris-base, HCl,
Acrylamide, Bisacrylamide, TEMED,
APS, Ultrapure water
4%
10%, 16%, 10–20%
2.6%
8.3
Zymogram Gels
Tris Glycine Gels with 4%
a substrate, casein or
No substrate
gelatin
10%, 12%, 4–16%
2.6%
8.6
IEF Gels
Acrylamide, BisNone
acrylamide, TEMED,
APS, Ultrapure water,
2% ampholytes
pH 3–7
2.6%
5.0
pH 3–10
6.0
TBE Gels
Tris-base, Boric acid,
EDTA, Acrylamide,
Bis-acrylamide,
TEMED, APS,
Ultrapure water
4%
6%, 8%, 10%, 20%,
4–12%, 4–20%
2.6%
8.3
TBE-Urea Gels
Tris-base, Boric acid,
EDTA, Acrylamide,
Bis-acrylamide,
TEMED, APS,
Ultrapure water,
7M Urea
4%
6%, 10%, 15%
3.8–5%
8.7
DNA Retardation
Gels
6% polyacrylamide
gels prepared with
half strength TBE gel
buffer
None
6%
2.6%
8.3
Important
Novex® Pre-Cast gels do not contain SDS. These gels can be used for nondenaturing (native) and denaturing gel electrophoresis.
For optimal and total separation ranges for each specific gel percentage, consult
the Gel Migration Charts on (page 72).
5
Gel Selection
Choosing a Gel for
Your Application
To obtain the best results, it is important to choose the correct gel percentage,
buffer system, gel format, and thickness for your application.
Review the following section, and Well Volume (page 8) to determine the type
of gel that is best suited for your application.
Refer to the Novex® Gel Migration Charts (see page 72) to find the gel with the
region of maximum resolution best suited for your sample. The leading protein
molecules should migrate about 70% of the length of gel for best resolution.
Protein Separation
Applications
Separation of proteins over a wide range of molecular weights
Use Novex® Tris-Glycine Gels for separating proteins over a wide molecular
weight range (6–200 kDa) under denaturing or non-denaturing conditions.
Resolve large molecules with low percentage gels, and small molecules with
high percentage gels. If the molecular weight of the molecule is unknown, or
the sample contains a wide range of molecules, use a gradient gel.
Separation of low molecular weight proteins and peptides
The Novex® Tricine Gels provide high resolution of low molecular weight
proteins and peptides (2–200 kDa). Tricine gels give the best results under
denaturing conditions.
Isoelectric focusing (IEF)
Use Novex® IEF Gels for native (vertical) IEF of proteins. The pH 3–10 gels have
a pI performance range of 3.5–8.5 and pH 3–7 gels have a pI performance range
of 3.0–7.0.
Protease detection
The Novex® Zymogram Gels are used for detecting and characterizing
proteases that utilize casein or gelatin as the substrate. Proteins are run under
denaturing conditions and then renatured for enzymatic activity.
2D separation of proteins
The ZOOM® Gels are specifically designed for second dimension
electrophoresis of 7.0 cm IPG strips. Gels with 2D wells can also be used, but
only accommodate IPG strips of 6.5 cm.
Continued on next page
6
Gel Selection, Continued
Nucleic Acid
Separation
Applications
Nucleic acid analysis
The Novex® Pre-Cast Gels are capable of resolving nucleic acids in the range of
10–3000 bp.
Novex® TBE Gels are used to perform analysis of DNA fragments from
restriction digest and PCR products, Southern analysis, and primer analysis.
Novex® TBE-Urea Gels are used for denaturing nucleic acid analysis and are
suited for RNase Protection Assays, in-vitro transcription studies, RNA stability
studies, and oligonucleotide purification.
Gel shift assays
The Novex® 6% DNA Retardation Gels are used to perform gel shift assays.
7
Well Volume
Recommended
Loading Volumes
The recommended loading volumes and protein load per band by the detection
method are provided in the table below.
Note: The 9-well gels are compatible with any eight-channel pipettors used for
loading samples from 96-well plates. An additional lane is included for loading
protein molecular weight standard.
Well Types
1 well
2D well
IPG well
5 well
9 well
10 well
12 well
15 well
Maximum Load
Volume
Coomassie Staining
Ethidium Bromide
Silver Staining
700 μL
12 μg/band
2.4 μg/band
1.0 mm
1.5 mm
400 μL
600 μL
12 μg/band
2.0 μg/band
1.0 mm
7 cm IPG Strip
N/A
N/A
Scale your
sample load for
the sensitivity of
your silver
staining kit.
1.0 mm
60 μL
2 μg
400 ng/band
1.0 mm
28 μL
0.5 μg/band
100 ng/band
1.0 mm
1.5 mm
25 μL
37 μL
0.5 μg/band
100 ng/band
1.0 mm
20 μL
0.5 μg/band
100 ng/band
1.0 mm
15 μL
0.5 μg/band
100 ng/band
1.5 mm
25 μL
1.0 mm
Choosing the
Appropriate Well
for Your
Application
8
Maximum Protein Load Per Band by Detection Method
For use with the
SilverQuest™ or
SilverXpress ®
Silver Staining
Kits, we
recommend a
protein load of
1 ng/band.
Choose the type of well for your application based upon the volume of your
sample. The more wells a comb has, and the thinner the gel is, the lower the
sample loading volume.
Note: Proteins transfer out of a 1.0 mm gel more easily than from a 1.5 mm gel.
Gel Staining
Staining Novex®
Pre-Cast Gels
The Novex® Pre-Cast Gels are compatible with most silver staining protocols.
We recommend using the SilverQuest™ Silver Staining Kit or the SilverXpress®
Silver Staining Kit (see pages 35–43) for silver staining of Novex® Gels.
Novex® Pre-Cast Gels are compatible with any of the standard Coomassie
staining procedures. Protocols that are accelerated by heat are preferable, as
heat can fix proteins (especially smaller peptides). The SimplyBlue™ SafeStain
(see page 36) and Novex® Colloidal Blue Staining Kit (see page 37) are
recommended for staining Novex® Gels.
Stain Type
Coomassie Blue
Sensitivity
100–500 ng
™
General
1 ng
Tris-Glycine, Bis-Tris,
Tricine, TBE
Low sample quantity,
Nucleic acid
0.3–2.5 ng
Bis-Tris, Tricine, TBE
8–16 ng
Colloidal Coomassie Blue
<10 ng
SimplyBlue SafeStain
SilverXpress
®
SilverQuest™
Application
Tris-Glycine, Bis-Tris,
Tricine, native
Coomassie Fluor Orange
™
Gel Type
Compatibility
5 ng
0.3–0.9 ng (50 bp)
SYPRO® Ruby
0.25–1 ng
Tris-Glycine, Bis-Tris,
Tricine, native
Low sample quantity,
Nucleic acid, Mass Spec
Pro-Q® Diamond
1–16 ng
Tris-Glycine, Bis-Tris
Phosphoprotein
Pro-Q Emerald
0.5–3 ng
Tris-Glycine
Glycoprotein
Ethidium Bromide
10 ng (50 bp)
TBE
Nucleic acid
SYBR® Green
60 pg (dsDNA)
TBE
Nucleic acid
®
100–300 pg (ssDNA)
1–2 ng (24 bp)
9
Methods
General Guidelines for Preparing Samples and Buffers
Introduction
The XCell SureLock™ Mini-Cell and a power supply are needed to perform
electrophoresis with Novex® Pre-Cast gels. Additional reagents supplied by the
user are described for each individual protocol.
General guidelines for preparing samples and buffers for Novex® Pre-Cast gels
are discussed below. Detailed instructions for preparing the sample buffer and
running buffer are described in the sections for each individual type of gel.
Recommended
Buffers
The recommended running buffer and sample buffer for each Novex® Pre-Cast
Gel is listed in the table below. Prepare your sample in the appropriate sample
buffer such that the final concentration of the sample buffer is 1X.
Running buffer must be diluted to 1X final concentration before use.
See page 63 for ordering information on pre-mixed buffers. See pages 65–71 for
recipes if you are preparing your own buffers.
Gel Type
Running Buffer
Sample Buffer
Novex Tris-Glycine
Gels (SDS-PAGE)
Tris-Glycine SDS Running Buffer (10X)
Tris-Glycine SDS Sample Buffer (2X)
Novex® Tris-Glycine
Gels (Native-PAGE)
Tris-Glycine Native Running Buffer
(10X)
Tris-Glycine Native Sample Buffer
(2X)
Novex® Tricine Gels
Tricine SDS Running Buffer (10X)
Tricine SDS Sample Buffer (2X)
Novex® Zymogram Gels
Tris-Glycine SDS Running Buffer (10X)
Tris-Glycine SDS Sample Buffer (2X)
IEF Gels
IEF Cathode Buffer (10X)
IEF Anode Buffer (50X)
IEF Sample Buffer (2X)
TBE Gels
TBE Running Buffer (5X)
Hi-Density TBE Sample Buffer (5X)
TBE-Urea Gels
TBE Running Buffer (5X)
TBE-Urea Sample Buffer (2X)
®
Prep TBE-Urea Sample Buffer (2X)
for preparative gels
DNA Retardation Gels
Reducing Agent
TBE Running Buffer (5X)
Hi-Density TBE Sample Buffer (5X)
When preparing samples for reducing gel electrophoresis, any of the following
reducing agents may be used:
 NuPAGE® Reducing Agent (see page 63 for ordering information)
 Dithiothreitol (DTT), 50 mM final concentration
 -mercaptoethanol, 2.5% final concentration

tris(2-carboxyethyl)phosphine (TCEP), 50 mM final concentration
Add the reducing agent to the sample up to an hour before loading the gel.
Avoid storing reduced samples for long periods, even if they are frozen.
Reoxidation of samples occur during storage and produce inconsistent results.
Continued on next page
10
General Guidelines for Preparing Samples and Buffers,
Continued
Running Reduced
and Non-Reduced
Samples
Heating Samples
For optimal results, we do not recommend running reduced and non-reduced
samples on the same gel.
If you do choose to run reduced and non-reduced samples on the same gel, do
not run reduced and non-reduced samples in adjacent lanes. The reducing agent
may have a carry-over effect on the non-reduced samples if they are in close
proximity.
Heating the sample at 100C in SDS containing buffer results in proteolysis
(Kubo, 1995). We recommend heating samples for denaturing electrophoresis
(reduced or non-reduced) at 85°C for 2–5 minutes for optimal results.
Do not heat the samples for non-denaturing (native) electrophoresis or
Zymogram Gels.
High Salt
Concentration in
Samples
High salt concentrations result in increased conductivity that affects protein
migration, and can result in gel artifacts in adjacent lanes containing samples
with normal salt concentrations. Perform dialysis or precipitate and resuspend
samples in lower salt buffer prior to electrophoresis.
Guanidine-HCl in
Samples
Samples solubilized in guanidine-HCl have high ionic strength, and produce
increased conductivity similar to high salt concentrations. In addition,
guanidine precipitates in the presence of SDS leading to various types of gel
artifacts. If possible, change the solubilization agent by dialysis prior to
electrophoresis.
Cell Lysates
Take the following considerations into account when performing
electrophoresis of cell lysates:

Genomic DNA in the cell lysate may cause the sample to become viscous
and affect protein migration patterns and resolution. Shear genomic DNA
to reduce viscosity before loading the sample.

Cells lysates contain soluble and insoluble fractions. The size of each
fraction depends upon the type of sample being analyzed. The nature of
the insoluble fraction may result in altered protein migration patterns and
resolution. Separate the two fractions by centrifugation and load them on
separate lanes for electrophoresis.

If RIPA buffer is used in cell lysis, subsequent blotting of proteins <40 kDa
may be inhibited due to the presence of Triton® X-100 in the buffer.
11
Tris-Glycine Gels
Tris-Glycine
Discontinuous
Buffer System
Novex® Tris-Glycine gels are based on the Laemmli System (Laemmli, 1970)
with minor modifications for maximum performance in the pre-cast format.
Unlike traditional Laemmli gels with a stacking gel pH of 6.8 and separating
gel pH of 8.8, Novex® Tris-Glycine gels have a pH of 8.65 for both regions.
The Tris-Glycine discontinuous buffer systems utilizes three ions:
Materials Supplied
by the User

Chloride () from the gel buffer serves as a leading ion due to its high
affinity to the anode relative to other anions in the system. The gel buffer
ions are Tris+ and Cl (pH 8.65).

Glycine () is the primary anion in the running buffer and serves as a
trailing ion. Glycine is partially negatively charged and trails behind the
highly charged chloride ions in the charged environment. The running
buffer ions are Tris+, Gly, and dodecylsulfate (pH 8.3).

Tris Base (+) is the common ion present in the gel buffer and running
buffer. During electrophoresis, the gel and buffer ions in the Tris-Glycine
system form an operating pH of 9.5 in the separation region of the gel.
The following reagents are needed to perform electrophoresis with Novex®
Tris-Glycine Gels. Ordering information for pre-mixed buffers is on page 63. If
you are preparing your own buffers, recipes are provided on pages 65–66.

Protein sample

Deionized water

Protein molecular weight markers
For denaturing electrophoresis

Novex® Tris-Glycine SDS Sample Buffer

NuPAGE® Reducing Agent

Novex® Tris-Glycine SDS Running Buffer
For non-denaturing (native) electrophoresis
Preparing Running
Buffer

Novex® Tris-Glycine Native Sample Buffer

Novex® Tris-Glycine Native Running Buffer
Use 1X Tris-Glycine SDS Running Buffer for electrophoresis of denatured
samples, or 1X Native Running Buffer for electrophoresis of native samples.
1.
Prepare 1,000 mL of Running Buffer as described below:
Reagent
Amount
®
10X Novex Tris-Glycine SDS or 10X Native Running Buffer
100 mL
Deionized Water
900 mL
Total Volume
2.
1,000 mL
Mix the buffer thoroughly and use it to fill the Upper and Lower Buffer
Chambers of the assembled XCell SureLock™ Mini-Cell for electrophoresis.
Continued on next page
12
Tris-Glycine Gels, Continued
Preparing Samples
for Denaturing
Electrophoresis
To separate proteins by mass alone, denature samples using SDS Sample Buffer
and heating.
1.
Prepare each sample as described below:
Reagent
Sample
Novex® Tris-Glycine SDS Sample Buffer (2X)
Deionized Water
Total Volume
2.
Amount
x μL
5 μL
to 5 μL
10 μL
Heat the sample at 85C for 2 minutes. Load the samples onto the gel
immediately.
Note: For reduced samples, add the reducing agent to a final concentration
of 1X immediately prior to electrophoresis to obtain the best results.
Preparing Samples
for Native
Electrophoresis
To separate proteins by charge:mass ratio in their native conformation, use
non-denaturing (native) electrophoresis.
1.
Prepare each sample as described below:
Reagent
Sample
Novex® Tris-Glycine Native Sample Buffer (2X)
Deionized Water
Total Volume
2.
Amount
x μL
5 μL
to 5 μL
10 μL
Load the samples onto the gel immediately. Do not heat samples for native
electrophoresis.
Electrophoresis
Conditions
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell. Run the gel at 125 V constant. See page 33 for additional
details on electrophoresis conditions.
Staining the Gel
Any of the techniques described on pages 35–46 are suitable for staining
Novex® Tris-Glycine Gels after electrophoresis.
13
Tricine Gels
Tricine Buffer
System
The Tricine system is a modification of the Tris-Glycine discontinuous buffer
system (see page 12) developed by Schaegger and von Jagow (Schaegger and
von Jagow, 1987) specifically designed for resolving peptides and low
molecular weight proteins.
In the Tris-Glycine system, proteins are stacked in the stacking gel between the
highly mobile leading chloride ion (in the gel buffer) and the slower trailing
glycine ion (in the running buffer). These stacked protein bands undergo
sieving once they reach the separating gel.
However, the resolution of smaller proteins (<10 kDa) is hindered by the
continuous accumulation of free dodecylsulfate (DS) ions (from the SDS sample
and running buffers) in the stacking gel. Smaller proteins mix with DS ions in
the zone of stacked DS micelles, resulting in fuzzy bands and decreased
resolution. The mixing also interferes with the fixing and staining of smaller
proteins.
To avoid this problem, the Tricine system uses a low pH gel buffer and replaces
the trailing glycine ion with a fast moving tricine ion in the running buffer. The
smaller proteins that previously migrated with the stacked DS micelles in the
Tris-Glycine system become well separated from DS ions in the Tricine system,
resulting in more efficient stacking and destacking of low molecular weight
proteins, sharper bands, and higher resolution
Advantages of
Tricine Gels
Materials Supplied
by the User
The Tricine Gels have the following advantages over the Tris-Glycine Gels for
resolving proteins in the molecular weight range of 2–20 kDa:

Allows resolution of proteins with molecular weights as low as 2 kDa

Ideal for direct sequencing of proteins after transferring to PVDF as tricine
does not interfere with sequencing

Minimizes protein modification because of a lower pH
The following reagents are needed to perform electrophoresis with Novex®
Tricine Gels. Ordering information for pre-mixed buffers is on page 63. If you
are preparing your own buffers, recipes are provided on page 67.

Protein sample

Deionized water

Protein molecular weight markers

Novex® Tricine SDS Sample Buffer

NuPAGE® Reducing Agent for reduced samples

Novex® Tricine SDS Running Buffer
Continued on next page
14
Tricine Gels, Continued
Preparing Running
Buffer
Use 1X Novex® Tricine SDS Running Buffer for electrophoresis of Tricine gels.
1.
Prepare 1,000 mL of Running Buffer as described below:
Reagent
Amount
®
Novex Tricine SDS Running Buffer (10X)
Deionized Water
100 mL
900 mL
Total Volume
2.
1,000 mL
Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer
Chambers of the XCell SureLock™ Mini-Cell for electrophoresis.
Novex® Tricine Gel are not compatible with buffers for Tris-Glycine gels.
Preparing Samples

Samples run in Tris-Glycine SDS Sample Buffer are poorly resolved.

Samples run in Tris-Glycine SDS Running Buffer take longer to complete
and result in poor resolution of smaller proteins.
Protein samples for Tricine Gels can be denatured, or denatured and reduced.
1.
2.
Prepare each reduced or non-reduced samples for running on Tricine gels
as described below:
Reagent
Reduced
Sample
Sample
Novex® Tricine SDS Sample Buffer (2X)
NuPAGE® Reducing Agent (10X)
Deionized Water
Total Volume
x μL
5 μL
1 μL
to 4 μL
10 μL
Non-reduced
Sample
x μL
5 μL
–
to 5 μL
10 μL
Heat samples at 85C for 2 minutes. Load the samples onto the gel
immediately.
Note: For reduced sample, add the reducing agent immediately prior to
electrophoresis to obtain the best results. Leave an empty lane between
samples with and without reducing agent to prevent diffusion of the
reducing agent into non-reduced sample lanes.
Electrophoresis
Conditions
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell. Run the gel at 125 V constant. See page 33 for additional
details on electrophoresis conditions.
Staining the Gel
Any of the techniques described on pages 35–46 are suitable for staining
Novex® Tricine Gels after electrophoresis.
15
Zymogram Gels
Zymogram
Technique
Zymogram analysis is used for detecting and characterizing
metalloproteinases, collagenases, and other proteases that can utilize casein or
gelatin as a substrate. Protease samples are denatured in SDS buffer under
non-reducing conditions and without heating, and run on a Zymogram Gel
using Tris-Glycine SDS Running Buffer. After electrophoresis, the enzyme is
renatured by incubating the gel in Zymogram Renaturing Buffer containing a
non-ionic detergent. The gels are then equilibrated in Zymogram Developing
Buffer (to add divalent metal cations required for enzymatic activity), and then
stained and destained. Regions of protease activity appear as clear bands
against a dark blue background where the protease has digested the substrate.
Types of
Zymogram Gels
Three different types of Zymogram Gels are available from Invitrogen. Details
are listed on the table below.
Gel Type
Separating Gel
®
Substrate
Sensitivity
–6
Novex Zymogram
Gelatin Gel
10% Tris-Glycine gel
with 0.1% gelatin
10 units of collagenase
Novex® Zymogram
Casein Gel
12% Tris-Glycine gel
-casein
7 × 10–4 units of trypsin
Novex® Zymogram Blue
Casein Gel
4–16% Tris-Glycine gel
blue-stained -casein
1.5 × 10–3 units of trypsin
Materials Supplied
by the User
Important
The following reagents are needed to perform electrophoresis with Novex®
Zymogram Gels. Ordering information for pre-mixed buffers is on page 63. If
you are preparing your own buffers, recipes are provided on pages 65–68.

Protein sample

Deionized water

Protein molecular weight markers

Novex® Tris-Glycine SDS Sample Buffer

Novex® Tris-Glycine SDS Running Buffer

Novex® Zymogram Renaturing Buffer

Novex® Zymogram Developing Buffer

Do not treat zymogram samples with reducing agents. Some proteases are
multiunit complexes that require the full subunit assembly for activity.

Load 2–3 times the recommended amount of unstained molecular weight
marker required for a Tris-Glycine Gel. The marker needs to stain intensely
to be visualized against the dark background of the Zymogram Gel.

Leave an empty lane between protein molecular weight markers containing
reducing agent and protease sample lanes to prevent diffusion of the
reducing agent into the protease lane.
Continued on next page
16
Zymogram Gels, Continued
Preparing Running
Buffer
Use 1X Novex® Tris-Glycine SDS Running Buffer for electrophoresis of protease
samples on Zymogram Gels.
1.
Prepare 1,000 mL of Running Buffer as follows:
Reagent
Amount
®
Novex Tris-Glycine SDS Running Buffer (10X)
100 mL
Deionized Water
900 mL
Total Volume
2.
Preparing Samples
1,000 mL
Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer
Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.
Prepared samples without reducing agents so that multiunit proteases migrate
as a single unit that can be renatured after electrophoresis.
1. Prepare each sample as described below:
Reagent
Amount
Sample
Novex® Tris-Glycine SDS Sample Buffer (2X)
Deionized Water
Total Volume
2.
x μL
5 μL
to 5 μL
10 μL
Load the samples onto the gel immediately. Do not heat samples for
Zymogram Gels.
Electrophoresis
Conditions
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell. Run the gel at 125 V constant. See page 33 for additional
details on electrophoresis conditions.
Detecting Protease
Activity
After completing electrophoresis, renature the enzyme and develop the
Zymogram Gels to detect protease activity.
Requirements for the volume of Zymogram Renaturing Buffer and Zymogram
Developing Buffer may vary, depending upon the size of your developing tray.
Preparing
Renaturing Buffer
Up to two mini-gels can be treated with every 100 mL of 1X Novex® Zymogram
Renaturing Buffer.
1.
Prepare 100 mL of Renaturing Buffer as described below:
Reagent
®
Novex Zymogram Renaturing Buffer (10X)
10 mL
Deionized Water
90 mL
Total Volume
2.
Amount
100 mL
Mix thoroughly before use.
Continued on next page
17
Zymogram Gels, Continued
Preparing
Developing Buffer
Up to two mini-gels can be treated with every 100 mL of 1X Novex® Zymogram
Developing Buffer:
1.
Prepare 100 mL of Developing Buffer as described below:
Reagent
Amount
®
Novex Zymogram Developing Buffer (10X)
10 mL
Deionized Water
90 mL
Total Volume
2.
100 mL
Mix thoroughly before use.
Note: Gels will be treated with Developing Buffer twice, so additional
buffer may be required, depending upon the size of the developing tray.
Developing
Zymogram Gels
1.
Remove the gel from the cassette, or remove the top gel plate, and allow the
gel to remain on the bottom gel plate for support.
2.
Incubate the gel in 1X Novex® Zymogram Renaturing Buffer for 30 minutes
at room temperature with gentle agitation.
3.
Decant the Zymogram Renaturing Buffer and add 1X Novex® Zymogram
Developing Buffer to the gel.
4.
Equilibrate the gel for 30 minutes at room temperature with gentle
agitation.
5.
Decant the Developing Buffer and add fresh 1X Novex® Zymogram
Developing Buffer to the gel.
6.
Incubate the gel at 37°C for at least 4 hours, or overnight for maximum
sensitivity. The incubation time can be reduced to 1 hour for concentrated
samples. The optimal result is determined empirically by varying the
sample load or incubation time.
Staining Zymogram Zymogram (Blue Casein) 4–16% gels do not require staining.
Gels
For non-pre-stained Zymogram gels, stain the gels with Colloidal Blue Staining
Kit or the SimplyBlue™ Safestain as described on pages 36–37.
Areas of protease activity appear as clear bands against a dark background.
18
IEF Gels
Isoelectric
Focusing (IEF)
Isoelectric focusing (IEF) is an electrophoretic technique for the separation of
proteins based on their pI. The pI is the pH at which a protein has no net charge
and thus, does not migrate further in an electric field.
IEF Gels are used to determine the isoelectric point (pI) of a protein and to
detect minor changes in the protein due to post-translational modifications
such as phosphorylation and glycosylation.
In IEF, proteins are applied to polyacrylamide gels (IEF Gels) or immobilized
pH gradient (IPG) strips containing a fixed pH gradient. As the protein sample
containing a mixture of proteins migrates through the pH gradient, individual
proteins are immobilized in the pH gradient as they approach their pI.
Novex® IEF Gels contain 5% polyacrylamide and are used for native
applications. The pH 3–10 gels have a pI performance range of 3.5–8.5 and the
pH 3–7 gels have a pI performance range of 3.0–7.0.
2D Electrophoresis
Proteins separated on IEF Gels are suitable for use in two-dimensional (2D)
electrophoresis using Novex® Tris-Glycine or NuPAGE® Gels with a 2D-well or
ZOOM® format to separate focused proteins by mass.
Two-dimensional (2D) gel electrophoresis is a powerful and sensitive technique
for separating and analyzing protein mixtures from biological samples. 2D gel
electrophoresis is performed in two consecutive steps:
1.
2.
First dimension separation of proteins using isoelectric focusing.
Proteins are separated based on their isoelectric point using IEF gels or IPG
strips.
Second dimension separation of proteins using SDS-PAGE.
Proteins are separated based on their molecular weight using denaturing
polyacrylamide gel electrophoresis.
The gel is stained after 2D electrophoresis to visualize the separated proteins,
or the proteins are blotted onto membranes. Protein spots can be excised from
the gel or membranes and subjected to further analyses such as mass
spectrometry or chemical microsequencing to facilitate protein identification.
Power
Considerations for
IEF
During IEF, proteins migrate in an electric field until a stable pH gradient is
formed and the proteins settle into their pI. A high finishing voltage is applied
to focus the proteins into narrow zones. High voltage cannot be used during
the initial stages of IEF as movement of carrier ampholytes generate excessive
heat.
To obtain the best results, IEF is typically performed by gradually increasing
the voltage, then maintaining the final focusing voltage for 30 minutes.
Alternatively, IEF can be performed at constant power, so the voltage will
increase as the current decreases.
Continued on next page
19
IEF Gels, Continued
Materials Supplied
by the User
Preparing Anode
Running Buffer
(Lower Buffer
Chamber)
The following reagents are needed to perform isoelectric focusing with Novex®
IEF Gels. Ordering information for pre-mixed buffers is on page 63. If you are
preparing your own buffers, recipes are provided on pages 68–69.

Protein sample

Deionized water

IEF markers

Novex®IEF Sample Buffer

Novex®IEF Cathode Buffer

Novex®IEF Anode Buffer

Fixing solution
Prepare 1X IEF Anode Buffer using Novex® IEF Anode Buffer (50X).
1.
Prepare 1,000 mL of IEF Anode Buffer as follows:
Reagent
®
Novex IEF Anode Buffer (50X)
Deionized Water
Total Volume
2.
Preparing Cathode
Running Buffer
(Upper Buffer
Chamber)
20 mL
980 mL
1,000 mL
Mix thoroughly. Use this buffer to fill the Lower Buffer Chamber of the
XCell SureLock™ Mini-Cell for electrophoresis.
Prepare 1X IEF Cathode Buffer using the appropriate Novex® IEF Cathode
Buffer pH 3–10 (10X) or pH 3–7 (10X)
1.
Prepare 200 mL of IEF Cathode Buffer as follows:
Reagent
Novex® IEF Cathode Buffer (10X)
2.
Preparing Sample
Amount
Amount
20 mL
Deionized Water
180 mL
Total Volume
200 mL
Mix thoroughly. Use this buffer to fill the Upper Buffer Chamber of the
XCell SureLock™ Mini-Cell for electrophoresis.
Samples for IEF Gels are prepared without SDS to avoid affecting the pI of the
protein. Reducing agents are also not recommended for the same reason.
1.
Prepare samples for IEF Gels as described below:
Reagent
Sample
Novex® IEF Sample Buffer pH 3–10 or pH 3–7 (2X)
Deionized Water
Total Volume
2.
Amount
x μL
5 μL
to 5 μL
10 μL
Load the sample immediately. Do not heat samples for IEF Gels.
Continued on next page
20
IEF Gels, Continued
Electrophoresis
Conditions
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell. Run the gel at 100 V constant for 1 hour, followed by
200 V constant for 1 hour, and finish with 500 V constant for 30 minutes. See
page 33 for additional details on electrophoresis conditions.
Fixing the Gel
Fixing the proteins in the IEF gel is recommended after electrophoresis. The
fixing step also helps to remove carrier ampholytes from the gel, resulting in
lower background after staining.
Fixing solution consists of 12% TCA, or 12% TCA wtih 3.5% sulfosalicylic acid.
1.
Prepare 500 mL of fixing solution as follows:
Reagent
Trichloroacetic Acid (TCA)
60.0 g
Sulfosalicylic Acid (optional)
17.5 g
Deionized Water
Total Volume
Staining IEF Gels
Amount
2.
Mix solution thoroughly.
3.
Fix gels for 30 minutes.
to 500 mL
500 mL
IEF gels can be stained by Coomassie or colloidal blue techniques, refer to
pages 35–38.
If using the SimplyBlue™ SafeStain, wash the gel extensively to remove traces of
TCA from the fixation process to avoid formation of precipitate in the gel.
2D SDS-PAGE with
IEF Gels
After staining the gel and documenting the results, proteins separated by pI can
be separated by mass.
We recommend using NuPAGE® Bis-Tris or Novex® Tris-Glycine Gels with a
2D-well, or ZOOM® Gels for 2D SDS-PAGE.
2D-wells can fit strips of 6.5 cm, while ZOOM® IPG-wells can fit strips of 7 cm.
Fixing and staining the IEF gel prior to performing second dimension SDSPAGE has the following advantages over other methods of storing IEF gels:

Indefinite storage without loss of resolution

Easy to manipulate as bands are visible

Confirms quality of first dimension IEF before proceeding to SDS-PAGE
Continued on next page
21
IEF Gels, Continued
Materials Supplied
by the User
Equilibrating the
Gel
In addition to the appropriate gel with a 2D-well or IPG-well, the following
reagents are needed to perform 2D gel electrophoresis with Novex® Gels.

20% Ethanol

Sample Buffer (depending on your gel type)

Running Buffer (depending on your gel type)

Filter Paper

NuPAGE® Sample Reducing Agent (optional)

Iodoacetamide (optional)
The SDS in the sample buffer and running buffer for SDS-PAGE strips the stain
from proteins and resolubilizes the proteins for migration during 2D
electrophoresis.
1.
Incubate the IEF gel in 100 mL 20% ethanol for 10 minutes.
2.
Cut out the desired lane (strip) from the IEF gel for SDS-PAGE.
3.
Incubate the strip in 2 mL 2X SDS sample buffer and 0.5 mL ethanol for
3–5 minutes. Aspirate the sample buffer and rinse with 1X Running Buffer.
4.
Proceed
Optional Procedure for Reduced Samples:
22
1.
Incubate the IEF gel in 100 mL 20% ethanol for 10 minutes.
2.
Cut out the desired lane (strip) from the IEF gel for SDS-PAGE.
3.
Incubate the strip in 2 mL 2X SDS sample buffer and 0.5 mL ethanol for
3–5 minutes. Aspirate the sample buffer and rinse with 1X Running Buffer.
4.
Prepare Reducing Solution by diluting 250 μL of the NuPAGE® Sample
Reducing Agent (10X) in 1.75 mL of 1X SDS Sample Buffer.
5.
Incubate the strip in Reducing Solution for 3–5 minutes. Decant the
Reducing Solution.
6.
Prepare 125 mM Alkylating Solution by adding 58 mg of fresh
iodoacetamide to 2.5 mL of 1X SDS Sample Buffer.
7.
Incubate the strip in Reducing Solution for 3–5 minutes.
8.
Decant the Alkylating Solution and proceed to 2D Separation of Proteins
on Novex® IEF Gels (next page).
IEF Gels, Continued
2D Separation of
Proteins on Novex®
IEF Gels
A protocol for separating proteins in an IEF gel strip by SDS-PAGE with the
XCell SureLock™ Mini-Cell is provided below.
1.
Fill the 2D or IPG-well with the appropriate 1X SDS Running Buffer.
2.
Trim the IEF strip to a length of 5.8–5.9 cm (for 2D-wells) or 6.3–6.4 cm (for
ZOOM® IPG-wells) such that the strip includes the pH regions containing
your proteins of interest.
3.
Transfer the IEF gel strip into the well of a 1.0 mm or 1.5 mm gel cassette as
follows:
 For 1.0 mm Thick Gels
Slide the strip into the well using a gel-loading tip. Avoid trapping airbubbles between the gel strip and the surface of the gel. Wet a piece of
thick filter paper (5.8 × 4 cm) in 1X SDS Running Buffer and use it to
push the IEF gel strip down so it makes contact with the surface of the
gel (see figure below). The paper should hold the IEF gel strip in place.
Filter Paper
MW
Std

SDS Gel
IEF Gel
Strip
For 1.5 mm Thick Gels
Wet two pieces of thin filter paper (5.8 × 4 cm) in 1X SDS Running
Buffer. Sandwich and the IEF gel strip with the filter paper, such that
the edge of the gel strip protrudes ~0.5 mm beyond the edge of the
paper (see figure below). Insert the sandwich into the well and push the
strip so it comes in contact with the gel. Avoid trapping air-bubbles
between the gel strip and the surface of the gel.
Filter Paper
Filter
Paper
IEF Gel Strip
Electrophoresis
Conditions
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell.
Run the gel at 125 V constant. After the dye front has moved into the stacking
gel (~10 min), disconnect the power supply, remove the filter paper, and
resume electrophoresis to completion.
Staining the Gel
Stain the gel with the appropriate method for the type of gel and sample
amount after electrophoresis. Refer to the techniques described on pages 35–46.
23
ZOOM® Gels
ZOOM® Gels
ZOOM® Gels are used for 2D analysis of proteins following isoelectric focusing
of IPG strips. ZOOM® Gels are 1.0 mm thick, and contain an IPG well and a
molecular weight marker well. The IPG well is designed to accommodate a
7.0 cm IPG strip.
Two types of ZOOM® Gels are available (see page 63 for ordering information)

NuPAGE® Novex® 4–12% Bis-Tris ZOOM® Gel

Novex® 4–20% Tris-Glycine ZOOM® Gel
2D Separation of
IPG Strips
The second dimension electrophoresis procedure involves reducing and
alkylating the proteins focused on your IPG strip in equilibration buffer,
loading the strip on your second dimension gel, and performing SDS-PAGE.
For 2D separation of Novex® IEF Gel strips, see page 21.
Materials Supplied
by the User
You will need the following items for running ZOOM® Gels (see pages 63–64
for ordering information on Invitrogen products):
Equilibrating the
IPG Strip

4X NuPAGE® LDS Sample Buffer

NuPAGE® Sample Reducing Agent

NuPAGE® Novex® 4–12% Bis-Tris ZOOM® Gel or Novex® 4–20% TrisGlycine ZOOM® Gel

Running Buffer (depending on your gel type)

0.5% agarose solution

Iodoacetamide

Plastic flexible ruler or thin weighing spatula

15 mL conical tubes

Water bath set at 55C or 65C

Protein molecular weight marker
1.
Dilute 4X NuPAGE® LDS Sample Buffer to 1X with deionized water.
2.
Add 500 μL of the NuPAGE® Sample Reducing Agent (10X) to 4.5 mL of the
1X NuPAGE® LDS Sample Buffer from Step 1 in a 15 mL conical tube. Place
one IPG strip in this conical tube for equilibration.
3.
Incubate for 15 minutes at room temperature. Decant the Reducing Solution.
4.
Prepare 125 mM Alkylating Solution by adding 116 mg of fresh
iodoacetamide to 5 mL of 1X NuPAGE® LDS Sample Buffer from Step 1.
5.
Add 5 mL of Alkylating Solution (from Step 4) to the conical tube containing
the IPG strip. Incubate for 15 minutes at room temperature.
6.
Decant the Alkylating Solution and proceed immediately to SDS-PAGE,
page 25.
Continued on next page
24
ZOOM® Gels, Continued
SDS-PAGE
Electrophoresis
Conditions
A protocol for separating proteins in an IPG strip by SDS-PAGE with ZOOM®
Gels and the XCell SureLock™ Mini-Cell is provided below.
1.
Prepare 0.5% agarose solution in the appropriate running buffer and keep it
warm (55–65C) until you are ready to use the agarose solution.
2.
Cut the plastic ends of the IPG strip flush with the gel. Do not cut off any
portions of the gel.
3.
Slide the IPG strip into the ZOOM® Gel well.
4.
If the molecular weight marker well is bent, straighten the well using a gelloading tip.
5.
Align the IPG strip properly in the ZOOM® Gel well using a thin plastic
ruler or a weighing spatula. Avoid trapping air bubbles between the strip
and the gel while sliding the strip into the well.
6.
Pour ~ 400 μL of 0.5% agarose solution into the ZOOM® Gel well to seal the
IPG strip in place. Make sure the agarose solution does not overflow into
the molecular weight marker well.
See page 32 for instructions on running Novex® Pre-Cast Gels using the XCell
SureLock™ Mini-Cell.
Note: Do not use the ZOOM® IPGRunner™ Core for electrophoresis of the
second dimension gel. You must use the Buffer Core supplied with the XCell
SureLock™ Mini-Cell.
Perform electrophoresis at 200 V for 40 minutes for NuPAGE® Novex® Bis-Tris
ZOOM® Gels or at 125 V for 90 minutes for Novex® Tris-Glycine ZOOM® Gels.
Staining the Gel
Stain the gel with the appropriate method for the type of gel and sample
amount after electrophoresis. Refer to the techniques described on pages 35–46.
25
TBE Gels
Introduction
Novex® polyacrylamide TBE Gels provide high-resolution analysis of
restriction digests and PCR products. The TBE Gels give sharp, intense bands
and provide separations of double-strand DNA fragments from 10–3,000 base
pairs.
Advantages of TBE
Gels
Using polyacrylamide gels for nucleic acid separation provides the following
advantages over agarose gels:
Materials Supplied
by the User
Preparing Running
Buffer

High resolution and sensitivity

Lower background staining

Requires less sample concentration and volume

Efficient blotting

Easy to extract DNA from the gel and does not interfere with enzymatic
reactions

Accurate and reproducible results
The following reagents are needed to perform gel electrophoresis with Novex®
TBE Gels. Ordering information for pre-mixed buffers is on page 63. If you are
preparing your own buffers, recipes are provided on pages 69–70.

DNA sample

Deionized water

Appropriate DNA markers

Novex® Hi-Density TBE Sample Buffer

Novex® TBE Running Buffer
Use 1X Novex® TBE Running Buffer to perform electrophoresis.
1.
Prepare 1,000 mL of Running Buffer as follows:
Reagent
®
Novex TBE Running Buffer (5X)
200 mL
Deionized Water
800 mL
Total Volume
2.
Amount
1,000 mL
Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer
Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.
Continued on next page
26
TBE Gels, Continued
Preparing Samples
Novex® TBE Gels require only ~10% of the amount of sample used on large gels
or agarose gels. Dilute your standards and samples to ~ 0.01 OD (0.2 μg/band)
to avoid overloading the gel.
1.
Prepare samples for TBE gels as described below:
Reagent
Amount
Sample
Novex® Hi-Density TBE Sample Buffer (5X)
Deionized Water
Total Volume
2.
x μL
2 μL
to 8 μL
10 μL
Load the samples immediately on the gel.
Electrophoresis
Conditions
See page 32 instructions for running TBE Gel using the XCell SureLock™ MiniCell. Run the gel at 200 V constant. See page 33 for additional details on
electrophoresis conditions.
Migration of the
Dye Fronts
The size of the DNA fragments visualized at the dye fronts of the different TBE
Gels is shown in the table below.
Gel Type
Dye Front*
Bromophenol Blue (dark blue) Xylene Cyanol (blue green)
6% TBE Gel
65 bp
250 bp
8% TBE Gel
25 bp
220 bp
10% TBE Gel
35 bp
120 bp
20% TBE Gel
15 bp
50 bp
4–12% TBE Gel
35 bp
400 bp
4–20% TBE Gel
25 bp
300 bp
*accuracy is  5 bp
Staining the Gel
Novex® TBE Gels can be stained by silver staining, ethidium bromide, and
SYBR® Green staining techniques after electrophoresis. Refer to pages 39–47 for
more information on these techniques.
27
TBE-Urea Gels
Introduction
Novex® denaturing polyacrylamide TBE-Urea Gels provide high resolution of
short single-strand oligonucleotides. The gels provide excellent resolution for
fast size and purity confirmations of DNA or RNA oligos from 20–600 bases.
The TBE-Urea Gels contain 7 M urea for maximum denaturation.
MEND
ION
AT
RECOM
Materials Supplied
by the User
Preparing Running
Buffer
The following reagents are needed to perform gel electrophoresis with Novex®
TBE-Urea Gels. Ordering information for pre-mixed buffers is on page 63. If
you are preparing your own buffers, recipes are provided on pages 69–70.

DNA or RNA sample

Deionized water

Appropriate DNA or RNA markers

Novex® TBE-Urea Sample Buffer

Novex® Prep TBE-Urea Sample Buffer (for preparative electrophoresis
only)

Novex® TBE Running Buffer
To obtain optimal results with TBE-Urea Gels, observe the following
recommendations:

Use RNase-free ultrapure water

Prior to loading samples, flush wells several times with 1X TBE Running
Buffer to remove urea

Load samples quickly and avoid allowing the gel to stand for long periods
of time after loading to prevent diffusion

Use Prep TBE-Urea Sample Buffer for preparative gel electrophoresis as
this buffer does not contain any marker dyes

Wear gloves and use dedicated equipment to prevent contamination

Avoid using buffers with formamide on TBE-Urea polyacylamide gels as it
will result in fuzzy bands
Use 1X Novex® TBE Running Buffer to perform electrophoresis.
1.
Prepare 1,000 mL of Running Buffer as follows:
2.
Reagent
Amount
Novex® TBE Running Buffer (5X)
200 mL
Deionized Water
800 mL
Total Volume
1,000 mL
3.
Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer
Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.
4.
Flush wells of the gel several times with 1X TBE Running Buffer to remove
urea from the wells prior to loading samples to obtain sharp bands.
Continued on next page
28
TBE-Urea Gels, Continued
Preparing Samples
Novex® TBE-Urea Gels require only ~10% of the amount of sample used on
large gels or agarose gels. Dilute your standards and samples to ~ 0.01 OD
(0.2 μg/band) to avoid overloading the gel.
1.
Prepare samples for TBE-Urea Gels as described below:
Reagent
Amount
Sample
Novex® TBE-Urea Sample Buffer (2X)
Deionized Water
Total Volume
x μL
5 μL
to 5 μL
10 μL
2.
Heat samples at 70C for 3 minutes to denature the samples.
3.
Load the samples immediately on the gel. If the samples are not used
immediately, place them on ice to prevent renaturation.
Electrophoresis
Conditions
See page 32 instructions for running TBE-Urea Gel using the XCell SureLock™
Mini-Cell. Run the gel at 180 V constant. See page 33 for additional details on
electrophoresis conditions.
Migration of the
Dye Fronts
The size of the single-strand DNA fragments visualized at the dye fronts of the
different TBE-Urea Gels is shown in the table below.
Gel Type
Dye Front*
Bromophenol Blue
(dark blue)
Xylene Cyanol (light blue)
6% TBE-Urea Gel
25 bases
110 bases
10% TBE-Urea Gel
20 bases
55 bases
15% TBE-Urea Gel
10 bases
40 bases
*accuracy is  5 bases
Staining the Gel
Novex® TBE-Urea Gels can be stained by silver staining, ethidium bromide, and
SYBR® Green staining techniques after electrophoresis. Refer to pages 35–48 for
more information on these techniques.
29
DNA Retardation Gels
Gel-Shift Assay
Novex® DNA Retardation Gels consist of 6% polyacrylamide prepared with
0.5X TBE as the gel buffer. The 6% gel provides good resolution of fragments in
the range of 60–2500 bp used for DNA retardation assays.
The gel shift assay is based on the fact that the movement of a DNA molecule
through a non-denaturing polyacrylamide gel is hindered when bound to a
protein molecule (Revzin, 1989). This technique is used to characterize
DNA/protein complexes. The 0.5X TBE buffer is sufficient for good
electrophoretic separation yet low enough to promote DNA/ protein
interactions.
Detection is performed with ethidium bromide staining of DNA or, for greater
sensitivity, with radiolabeling the DNA or protein.
Materials Supplied
by the User
Preparing Samples
The following reagents are needed to perform gel electrophoresis with Novex®
DNA Retardation Gels. Ordering information for pre-mixed buffers is on page
63. If you are preparing your own buffers, recipes are provided on pages 69–70.

DNA sample

Deionized water

Novex® Hi-Density TBE Sample Buffer

Novex® TBE Running Buffer
1.
Prepare samples for DNA Retardation Gels as described below:
Reagent
Sample
Novex® Hi-Density TBE Sample Buffer (5X)
Deionized Water
Total Volume
2.
Amount
x μL
1 μL
to 9 μL
10 μL
Load the samples immediately on the gel.
Specific buffer conditions may be required during incubation of the protein and
DNA target sequence in order to minimize non-specific DNA/protein
interactions for certain samples.
If salt concentration is low (0.1 M or less), the samples can usually be loaded in
the incubation buffer after adding about 3–5% glycerol and a small amount of
bromophenol blue tracking dye.
Continued on next page
30
DNA Retardation Gels, Continued
Preparing Running
Buffer
Prepare 1,000 mL of 0.5X Novex® TBE Running Buffer as follows:.
1.
Reagent
®
Novex TBE Running Buffer (5X)
100 mL
Deionized Water
900 mL
Total Volume
2.
Amount
1,000 mL
Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer
Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.
Electrophoresis
Conditions
See page 32 instructions for running DNA Retardation Gels using the XCell
SureLock™ Mini-Cell. Run the gel at 100 V constant. See page 33 for additional
details on electrophoresis conditions.
Staining the Gel
Gel-shift assays use labeled (radioactive, fluorescent, biotin) DNA fragments
for visualization of results. Use the appropriate technique to develop the image
for the type of label you are using.
31
Electrophoresis of Novex® Pre-Cast Gels
Introduction
Instructions are provided below for electrophoresis of Novex® Pre-Cast Gels
using the XCell SureLock™ Mini-Cell. For more information on the XCell
SureLock™ Mini-Cell, refer to the manual (IM-9003). This manual is available on
our website at www.invitrogen.com or contact Technical Support (see page 76).
For information on sample and buffer preparation for Novex® Pre-Cast Gels,
see pages 10–31.
Protocol using
XCell SureLock™
Mini-Cell
Wear gloves and safety glasses when handling gels.
XCell SureLock™ Mini-Cell requires 200 mL for the Upper Buffer Chamber and
600 mL for the Lower Buffer Chamber.
1.
Remove the Novex® Pre-Cast Gel from the pouch.
2.
Rinse the gel cassette with deionized water. Peel off the tape from the
bottom of the cassette.
3.
Gently pull the comb out of the cassette in one smooth motion.
4.
Rinse the sample wells with the appropriate 1X Running Buffer. Invert the
gel and shake the gel to remove the buffer. Repeat two more times.
5.
Orient the two gels in the Mini-Cell such that the notched “well” side of
the cassette faces inwards toward the Buffer Core. Seat the gels on the
bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.
Refer to the XCell SureLock™ Mini-Cell manual (IM-9003) for detailed
instructions.
Note: If you are running just one gel, use the plastic Buffer Dam in place of
the second gel cassette to form the Upper Buffer Chamber.
6.
Fill the Upper Buffer Chamber with a small amount of the Running Buffer
to check for tightness of seal. If you detect a leak from Upper to the Lower
Buffer Chamber, discard the buffer, reseal the chamber, and check the seal
again.
7.
Once the seal is tight, fill the Upper Buffer Chamber (Inner) with the
appropriate 1X Running Buffer. The buffer level must exceed the level of
the wells.
8.
Load an appropriate volume of sample at the desired protein
concentration onto the gel (see page 8 for recommended loading volumes).
9.
Load appropriate protein molecular weight markers (see page 64 for
ordering information).
10. Fill the Lower Buffer Chamber with 600 mL of the appropriate 1X Running
Buffer.
11. Place the XCell SureLock™ Mini-Cell lid on the Buffer Core. With the power
on the power supply turned off, connect the electrode cords to the power
supply [red to (+) jack, black to (–) jack].
12. See next page for Electrophoresis Conditions.
Continued on next page
32
Power Supply Settings for Novex® Pre-Cast Gels
Electrophoresis
Conditions
Gel Type
Run your gels according to the following protocol:
Voltage
Tris-Glycine Gels 125 V constant
(SDS-PAGE)
Expected
Current*
Run Time
Start: 30–40mA
90 minutes (dependent on gel type)
End: 8–12 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Start: 6–12 mA
1–12 hours
Tris-Glycine Gels 125 V constant
(Native-PAGE)
End: 3–6 mA
Tricine Gels
Start: 80 mA
90 minutes (dependent on gel type)
End: 40 mA
Run the gel until the phenol red tracking
dye reaches the bottom of the gel.
Start: 30–40 mA
90 minutes (dependent on gel type)
End: 8–12 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Zymogram Gels
IEF Gels
125 V constant
125 V constant
100 V constant: 1 hour Start: 5 mA
2.5 hours
200 V constant: 1 hour End: 6 mA
500 V constant: 30 min
TBE Gels
200 V constant**
6% TBE-Urea
Gels
180 V constant**
10% TBE-Urea
Gels
180 V constant**
15% TBE-Urea
Gels
180 V constant**
DNA
Retardation Gels
100 V constant
Start: 10–18 mA
30–90 minutes (dependent on gel type)
End: 4–6 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Start: 19 mA
50 minutes
End: 14 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Start: 15 mA
60 minutes
End: 8 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Start: 13 mA
75 minutes
End: 6 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
Start: 12–15 mA
90 minutes
End: 6–15 mA
Run the gel until the bromophenol blue
tracking dye reaches the bottom of the gel.
*Expected start and end current values are stated for single gels.
**Voltages up to 250 V may be used to reduce the run time.
33
Opening Novex® Pre-Cast Gel Cassettes
Removing the Gel
after
Electrophoresis
1.
After electrophoresis is complete, shut off the power, disconnect electrodes,
and remove gel(s) from the XCell SureLock™ Mini-Cell.
2.
Separate each of the three bonded sides of the cassette by inserting the Gel
Knife into the gap between the two plastic plates that make up the cassette.
The notched (“well”) side of the cassette should face up.
3.
Push down gently on the knife handle to separate the plates. Repeat on
each side of the cassette until the plates are completely separated.
Caution: Use caution while inserting the Gel Knife between the two plates
to avoid excessive pressure on the gel.
4.
Carefully remove and discard the top plate, allowing the gel to rest on the
bottom (slotted) plate.
5.
If blotting, proceed to page 52 without removing the gel from the bottom
plate.
6.
If staining, remove the gel from the plate by one of the methods:
7.
34

Use the sharp edge of the Gel Knife to remove the gel foot from the
bottom of the gel. Hold the Gel Knife at a 90° angle, perpendicular to
the gel and the slotted half of the cassette. Push down on the knife, and
then repeat the motion across the gel to cut off the entire foot. Hold the
plate and gel over a container with the gel facing downward and use
the knife to carefully loosen one lower corner of the gel and allow the
gel to peel away from the plate.

Hold the plate and gel over a container with the gel facing downward.
Gently push the Gel Knife through the slot in the cassette, until the gel
peels away from the plate. Cut the gel foot off of the gel after fixing and
staining, but before drying.
Fix and stain the gel as described on pages 35–48. For developing the
Zymogram gel for enzyme activity, see page 17. For fixing IEF gels, see
page 21.
Coomassie Staining
Introduction
Instructions are provided below for Coomassie staining Tris-Glycine,
Zymogram, IEF, and Tricine Gels using the SimplyBlue™ SafeStain, Colloidal
Blue Staining Kit, and Coomassie R-250.
If you are using other types of Coomassie staining kits, follow the appropriate
manufacturer’s recommendations.
If you are staining low molecular weight peptides (< 2.5 kDa), we recommend
fixing the gel in 5% glutaraldehyde and 50% methanol for one hour and then
follow the instructions in the Colloidal Blue Staining Kit Manual (IM-6025) for
small peptides.
Molecular Weight
Calibration
Guidelines and apparent molecular weight values for Novex® protein
molecular weight standards are provided on page 59.
Materials Supplied
by the User
You will need the following items for staining your gel (see page 63 for
ordering information on Invitrogen products):

Staining container

Deionized water

Orbital Shaker
For SimplyBlue™ SafeStain (see page 36):

SimplyBlue™ SafeStain

Optional: 20% NaCl

Optional: Microwave oven

12% Trichloroacetic acid (for IEF gels)
For Colloidal Blue Staining Kit (see page 37):

Colloidal Blue Staining Kit

Methanol

Optional: 20% Ammonium sulfate

Fixing solutino (for IEF gels)
For Coomassie R-250 Staining (see page 38):

0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid

Destaining Solution consisting of 10% ethanol and 7.5% acetic acid

Optional: Microwave oven
Continued on next page
35
Coomassie Staining, Continued
SimplyBlue™
SafeStain Protocol
The Basic Protocol for staining Novex® Gels with SimplyBlue™ SafeStain is
provided below. For the Microwave Protocol and staining large format gels,
refer to the SimplyBlue™ SafeStain Manual (IM-6050). This manual is available
on our website at www.invitrogen.com or contact Technical Support (page 76).
For general use with 1.0 mm and 1.5 mm thick Tris-Glycine Gels, and 1.0 mm
thick Tricine, Zymogram, and IEF Gels (8 cm × 8 cm).
After electrophoresis follow the instructions below. Be sure the mini-gel moves
freely in water or stain to facilitate diffusion during all steps.
Note: Stain Zymogram Gels with SimplyBlue™ SafeStain after renaturing and
developing the gel for enzyme activity.
1.
Fix IEF Gels in 100 mL 12% TCA for 15 minutes. The fixing step is not
required for Tris-Glycine, Tricine, and Zymogram Gels.
2.
Rinse the mini-gel 3 times for 5 minutes with 100 mL deionized water to
remove SDS and buffer salts, which interfere with binding of the dye to
the protein. Discard each rinse.
3.
Stain the mini-gel with enough SimplyBlue™ SafeStain (20–100 mL) to
cover the gel. Stain for 1 hour at room temperature with gentle shaking.
Bands will begin to develop within minutes. After incubation, discard the
stain. Stain cannot be re-used.
Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity
will decrease. If you need to leave the gel overnight in the stain, add 2 mL
of 20% NaCl (w/v) in water for every 20 mL of stain. This procedure will
not affect sensitivity.
4.
Wash the mini-gel with 100 mL of water for 1–3 hours. The gel can be left
in the water for several days without loss of sensitivity. There is a small
amount of dye in the water that is in equilibrium with the dye bound to
the protein, so proteins will remain blue.
5.
To obtain the clearest background for photography, perform a second
1 hour wash with 100 mL water.
Note: Sensitivity decreases at this point if the gel is allowed to stay in
the water more than 1 day. Reduction of free dye in the water favors
dissociation of the dye from the protein. If you need to store the gel in
water for a few days, add 20 mL of 20% NaCl.
6.
For gel drying, see page 49.
Continued on next page
36
Coomassie Staining, Continued
Colloidal Blue
Staining Kit
Protocol
A brief staining protocol for staining Novex® Gels with the Colloidal Blue
Staining Kit is provided below. For more details on the staining procedure,
refer to the Manual (IM-6025). This manual is available on our website at
www.invitrogen.com or contact Technical Support (see page 76).
1.
Fix the IEF Gel in fixing solution as described on page 21. This step is not
required for Tris-Glycine, Tricine, and Zymogram Gels.
2.
Prepare staining solution for a single gel as described in the table below.
For two gels, double the volume of reagents used for staining. Be sure to
shake Stainer B prior to making the solution.
Solutions
3.
Tris-Glycine, Tricine, and
Zymogram Gel
IEF Gel
Deionized Water
55 mL
58 mL
Methanol
20 mL
20 mL
Stainer B
5 mL
2 mL
Stainer A
20 mL
20 mL
Incubate the gel in this staining solution as follows at room temperature
with gentle shaking:

Tris-Glycine, Tricine, and Zymogram Gels for a minimum of 3 hours
and a maximum of 12 hours .

IEF Gels for 30 minutes.
4.
Decant staining solution and add a minimum of 200 mL of deionized water
per gel to the staining container. Gently shake gel in water for at least
7 hours. Gel will have a clear background after 7 hours in water.
5.
For gel drying, see page 49.
Note: Novex® Gels can be left in deionized water for up to 3 days without
significant change in band intensity and background clarity.
For long-term storage (over 3 days), keep the gel in a 20% ammonium sulfate
solution at 4°C.
Continued on next page
37
Coomassie Staining, Continued
Coomassie R-250
Staining Protocol
The Coomassie staining protocol described below is recommended for staining
Novex® Gels. You may use any Coomassie staining protocol of choice.
1.
Prepare the staining solution containing 0.1% Coomassie R-250 in
40% ethanol, 10% acetic acid.
2.
After electrophoresis, incubate 1 or 2 gels in a staining container containing
100 mL Coomassie Blue R-250 staining solution.
Caution: Use caution while performing the following steps using a
microwave oven. Do not overheat the staining solutions.
3.
Loosely cover the staining container and heat in a microwave oven at full
power for 1 minute. To prevent hazardous, flammable vapors from
forming, do not allow the solution to boil.
4.
Remove the staining container from the microwave oven and gently shake
the gel for 15 minutes at room temperature on an orbital shaker.
5.
Decant the stain and rinse the gel once with deionized water.
6.
Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.
7.
Place one or two stained gels in a staining container containing the 100 mL
destain solution.
8.
Loosely cover the staining container and heat in a microwave oven at full
power for 1 minute.
9.
Gently shake the gel at room temperature on an orbital shaker until the
desired background is achieved.
10. For gel drying, see page 49.
38
Silver Staining
Introduction
Instructions are provided below for silver staining Novex® Gels using the
SilverQuest™ Silver Staining Kit and the SilverXpress® Silver Staining Kit (see
page 63 for ordering information).
If you are using any other silver staining kit, follow the manufacturer’s
recommendations.
Molecular Weight
Calibration
Guidelines and apparent molecular weight values for Novex® protein
molecular weight standards are provided on page 64.
Materials Supplied
by the User
You will need following items for silver staining your gel (see page 63 for
ordering information on Invitrogen products):

Staining container

Rotary Shaker

Ultrapure water (>18 megohm/cm resistance recommended)

Teflon coated stir bars

Disposable 10 mL pipettes

Clean glass bottles for reagent preparation

Graduated glass cylinders

Protein molecular weight markers (Mark 12™ Unstained Standard,
recommended)
For SilverQuest™ Staining:

SilverQuest™ Silver Staining Kit

30% ethanol (made with ultrapure water)

100% ethanol

Fixative (40% ethanol, 10% acetic acid, made with ultrapure water)
For SilverXpress® Staining:

SilverXpress® Silver Staining Kit

Methanol

Acetic acid

Sulfosalicylic acid

Trichloroacetic acid (TCA)
Continued on next page
39
MEND
ION
AT
RECOM
Silver Staining, Continued
Preparing
Solutions for
SilverQuest™ Silver
Staining
For optimal silver staining results, follow these guidelines:

Be sure to wear clean gloves that have been rinsed with deionized water
while handling gels

Use clean containers and designate these containers for silver staining
purposes only

Make sure the size of the container permits free movement of the gel
during shaking and complete immersion in solution while staining

Do not touch the gel with bare hands or metal objects and do not put
pressure on gels while handling or changing solutions

Use teflon coated stir bars and clean glass containers to prepare reagents

Avoid cross contamination of kit reagents

Use freshly made solutions
Use the reagents provided in the SilverQuest™ Silver Staining Kit to prepare the
following solutions for staining:

Sensitizing solution
Ethanol
30 mL
Sensitizer
10 mL
Ultrapure water

Staining solution
Stainer
Ultrapure water

to 100 mL
1 mL
to 100 mL
Developing solution
Developer
10 mL
Developer enhancer
1 drop
Ultrapure water
to 100 mL
Note: You may prepare all solutions immediately before starting the staining
protocol or prepare them as you proceed to the next step.
Continued on next page
40
Silver Staining, Continued
SilverQuest™
Microwave Silver
Staining Protocol
The Fast Staining protocol (using a microwave oven) for silver staining Novex®
Gels using SilverQuest™ Silver Staining Kit is described below. For the Basic
Protocol and more details on the staining procedure, refer to the SilverQuest™
Silver Staining Kit Manual (IM-6070). This manual is available on our website at
www.invitrogen.com or contact Technical Support (see page 76).
Use 100 mL of each solution for each 1.0 mm thick, 8 × 8 cm Novex® Gel.
Note: You may have to optimize the staining protocol, if the dimensions of your
gel are not the same as mentioned above.
Caution: Use caution while performing the Fast Staining Protocol using a
microwave oven. Do not overheat the staining solutions.
1. After electrophoresis, place the gel in a clean microwaveable staining tray of
the appropriate size. Rinse the gel briefly with ultrapure water.
2. Place the gel in 100 mL of fixative and microwave at high power (700 watts)
for 30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature. Decant the fixative.
3.
Wash the gel with 100 mL of 30% ethanol in a microwave at high power for
30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature on a rotary shaker. Decant the ethanol.
4.
Add 100 mL of Sensitizing solution to the washed gel. Microwave at high
power for 30 seconds. Remove the gel from the microwave and place it on a
rotary shaker for 2 minutes at room temperature. Decant the Sensitizing
solution.
5.
Add 100 mL ultrapure water to the gel. Microwave at high power for
30 seconds. Remove the gel from the microwave and gently agitate it for
2 minutes at room temperature. Decant the water, and repeat the step one
more time.
6.
Place the gel in 100 mL of Staining solution. Microwave at high power for
30 seconds. Remove the gel from the microwave and gently agitate it for
5 minutes at room temperature.
7.
Decant the Staining solution and wash the gel with 100 mL of ultrapure
water for 20–60 seconds. Do not wash the gel for more than a minute.
8.
Place the gel in 100 mL of Developing solution and incubate for 5 minutes at
room temperature with gentle agitation on a rotary shaker. Do not
microwave.
9.
Once the desired band intensity is achieved, immediately add 10 mL of
Stopper directly to the gel still immersed in Developing solution and gently
agitate the gel for 10 minutes. The color changes from pink to clear
indicating the end of development.
10. Wash the gel with 100 mL of ultrapure water for 10 minutes. For gel drying,
see page 49.
If you need to destain the gel for mass spectrometry analysis, see the
SilverQuest™ Silver Staining Kit Manual (IM-6070).
Continued on next page
41
Silver Staining, Continued
Preparing
Solutions for
SilverXpress®
Silver Staining
Prepare the reagents as described below. If you are staining two gels, double
the reagent volumes.

Fixing solution for Tris-Glycine and Tricine Gels
Methanol
100 mL
Acetic Acid
20 mL
Ultrapure water
to 200 mL

Fixing solution for TBE, TBE-Urea Gels
Sulphosalicylic acid
7g
TCA
24 g
Ultrapure water
to 200 mL

Sensitizing solution
Methanol
Sensitizer
Ultrapure water


100 mL
5 mL
to 200 mL
Staining solution
Stainer A
Stainer B
Ultrapure water
5 mL
5 mL
90 mL
Developing Solution
Developer
Ultrapure water
5 mL
95 mL
Continued on next page
42
Silver Staining, Continued
SilverXpress®
Silver Staining
Protocol
The following staining procedure is for 1 mm thick Novex® Gels. If you are
using 1.5 mm thick Novex® Gels, double the incubation time.
For gel drying, see page 49.
Note: Gels may be stored in the second Sensitizing Solution overnight, if
desired.
Step
Solution
Vol/Gel
Gel Type
Tris-Glycine
1A
Fix the gel in Fixing Solution.
Tricine
TBE/TBE-Urea
IEF
200 mL
10 minutes
10 minutes 10 minutes
10 minutes
N/A
N/A
N/A
10 minutes
100 mL
10 minutes
30 minutes 10 minutes
30 minutes
100 mL
10 minutes
30 minutes 10 minutes
30 minutes
Decant the Sensitizing
Solution and rinse the gel
twice with ultrapure water.
200 mL
5 minutes
5 minutes
5 minutes
5 minutes
200 mL
5 minutes
5 minutes
5 minutes
5 minutes
4
Incubate the gel in Staining
Solution.
100 mL
15 minutes
15 minutes 30 minutes
15 minutes
5A
Decant the Staining Solution
and rinse the gel twice with
ultrapure water.
200 mL
5 minutes
5 minutes
5 minutes
5 minutes
200 mL
5 minutes
5 minutes
5 minutes
5 minutes
6
Incubate the gel in
Developing Solution.
100 mL
3–15 minutes 3–15
minutes
3–15 minutes
3–15
minutes
7
Add the Stopping Solution
directly to the gel when the
desired staining intensity is
reached.
5 mL
10 minutes
10 minutes 10 minutes
10 minutes
Decant the Stopping Solution
and wash the gel three times
in ultrapure water.
200 mL
10 minutes
10 minutes 10 minutes
10 minutes
200 mL
10 minutes
10 minutes 10 minutes
10 minutes
200 mL
10 minutes
10 minutes 10 minutes
10 minutes
1B
2A
2B
3A
3B
5B
8A
8B
8C
Decant the Fixing Solution
and incubate the gel in two
changes of Sensitizing
Solution.
N/A
43
SYPRO® Ruby Staining
Introduction
Instructions are provided below for a basic and rapid protocol for Novex® PreCast Gels (Novex® Tris-glycine gels, Novex® Tricine gels, ZOOM® gels, and
Novex® IEF gels) for the detection of proteins, including glycoproteins and
phosphoproteins.
Advantages of
SYPRO® Ruby
Staining
SYPRO® Ruby provides the following advantages:

Linear quantitation range of over three orders of magnitude

Compatible with subsequent analysis of proteins by Edman based
sequencing or mass spectrometry in 1D or 2D format

Compatible with non-denaturing gels and IEF gels (basic protocol)
Guidelines and apparent molecular weight values for Novex® protein
molecular weight standards are provided on page 64.
Materials Supplied
by the User
You will need following items for silver staining your gel (see page 63 for
ordering information on Invitrogen products):
MEND
ION
AT
RECOM
Molecular Weight
Calibration

SYPRO® Ruby gel stain

Staining containers, 1 per gel (see below for details)

Reagent-grade methanol

Reagent-grade glacial acetic acid

Trichloroacetic acid (for IEF gels only)

Ultrapure water (18 megohm-cm recommended)

Rotary shaker

Powder-free latex or vinyl gloves

Microwave oven (700–1200 W) (optional)

Water bath set at 80°C (optional)
General considerations for the protocol include the following:

Perform all fixation, staining, and washing steps with continuous, gentle
agitation (e.g., on an orbital shaker at 50 rpm).

We recommend polypropylene or polycarbonate containers for staining.
Glass dishes are not recommended. Staining containers should be
meticulously clean to minimize contamination and other artifacts.

For convenience, gels may be left in fix solution overnight or longer.

For convenience, gels may be left in SYPRO® Ruby stain indefinitely
without overstaining, although speckling artifacts tend to increase over
time.

As with any fluorescent stain, cover the gel container during staining and
subsequent wash steps to exclude light.
Continued on next page
44
SYPRO® Ruby Staining, Continued
Preparing
Solutions for
SYPRO® Ruby
Staining
Prepare the reagents as described below. If you are staining two gels, double
the reagent volumes. Increase volumes 1.5-fold for 1.5mm thick gels.



SYPRO® Ruby
Basic Protocol
Fix Solution
Methanol
Glacial Acetic Acid
Ultrapure water
100 mL
14 mL
to 200 mL
Fix Solution for IEF Gels
Methanol
Trichloroacetic Acid
Ultrapure water
40 mL
10 g
to 100 mL
Wash Solution
Methanol
Glacial Acetic Acid
Ultrapure water
10 mL
7 mL
to 100 mL
The basic protocol results in the maximum signal strength and widest linear
dynamic range for staining of denaturing gels, non-denaturing gels, and IEF
gels. Sensitivity is in the 1 ng range for most proteins.
1.
After electrophoresis, place the gel into a clean container with 100 mL of Fix
Solution and agitate on an orbital shaker for 30 minutes. Pour off the used
fix solution and repeat once more with fresh Fix Solution.
Note: For IEF Gels, place the gel into a clean container with 100 mL of IEF
Fix Solution and agitate on an orbital shaker for 3 hours. After fixing,
perform 3 washes in ultrapure water for 10 minutes each, before
proceeding to the staining step.
2.
Pour off the used fix solution.
3.
Add 60 mL of SYPRO® Ruby gel stain to the tray containing the gel. Agitate
on an orbital shaker overnight.
4.
Transfer the gel to a clean container and wash in 100 mL of Wash Solution
for 30 minutes. The transfer step helps minimize background staining
irregularities and stain speckles on the gel.
5.
Rinse the gel in ultrapure water for 5 minutes. Repeat the rinse a minimum
of one more time to prevent possible corrosive damage to your imager.
Note: If you are staining two gels, double the reagent volumes. Increase
volumes 1.5-fold for 1.5mm thick gels.
Visualization of
SYPRO® Ruby
Stained Gels
Proteins stained with SYPRO® Ruby protein gel stain are readily visualized
using a UV or blue-light source. The use of a photographic camera or CCD
camera and the appropriate filters is essential to obtain the greatest sensitivity.
Continued on next page
45
SYPRO® Ruby Staining, Continued
Using SYPRO®
Ruby Stain as a
Post-Stain
SYPRO® Ruby stain can be used to post-stain gels stained with other gel stains
such as Pro-Q® Diamond phosphoprotein gel stain, Pro-Q®Emerald 300
glycoprotein gel stain, Pro-Q® Sapphire or InVision™ oligohistidine-tag gel
stains, or Pro-Q® Amber transmembrane protein gel stain.
Always use SYPRO® Ruby stain last, as the SYPRO® Ruby signal can dominate
the signal from other stains. SYPRO® Ruby stain does not work well as a poststain for colorimetric stains such as Coomassie and silver stains.
46
SYBR® Green Staining
MEND
ION
AT
RECOM
Introduction
Procedure
Visualization of
SYBR® Green I
Stained Gels
The SYBR® Green I nucleic acid gel stain is a sensitive stain that can be used to
detect DNA in Novex® TBE and TBE-Urea Gels. As little as
20–60 pg of double stranded DNA, 100–300 pg of single stranded DNA, or
1–2 ng of a synthetic 24-mer can be detected, depending upon the wavelength
of transillumination.
General considerations for the protocol include the following:

We recommend polypropylene containers for staining. Glass dishes are
not recommended. Staining containers should be meticulously clean to
minimize contamination and other artifacts.

SYBR® Green I reagent has optimal sensitivity at pH 7.5–8.0.

For convenience, gels may be left in SYBR® Green I stain for up to 24 hours
with little decrease in sensitivity.
Perform post-staining of DNA on TBE or TBE-Urea Gels as follows:
1.
Prepare a 1:10,000 dilution of SYBR® Green I reagent in TE (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0), TBE, or TAE buffer.
2.
Remove the gel from the cassette using a Gel Knife, and place it in a
polypropylene staining container.
3.
Cover the gel with staining solution and incubate at room temperature for
10–40 minutes with gentle agitation. Protect the staining container from
light by covering it with aluminum foil.
SYBR® Green I stain is compatible with a wide variety of gel reading
instruments, ranging from ultraviolet transilluminators to argon laser and
mercury-arc lamp excitation gel scanners. SYBR® Green I stain is maximally
excited at 497 nm, but also has secondary excitation peaks at ~290 nm and ~380
nm. The fluorescence emission of SYBR® Green I stain bound to DNA is
centered at 520 nm.
47
Ethidium Bromide Staining
Introduction
A brief protocol is provided below for staining nucleic acids on TBE and TBEUrea Gels with ethidium bromide.
Procedure
Caution: Ethidium bromide is a powerful mutagen and is moderately toxic.
Wear gloves and protective clothing when handling ethidium bromide
solutions.
1.
Prepare 2 μg/ml solution of ethidium bromide in ultrapure water.
2.
Remove the gel from the cassette using a Gel Knife, and place it in a staining
container.
3.
Incubate the gel in the ethidium bromide solution for 20 minutes.
4.
Destain the gel by rinsing the gel three times with ultrapure water for
10 minutes.
Ethidium bromide staining of polyacrylamide gels requires at least 10 ng of
DNA for detection due to the quenching of the fluorescence by polyacrylamide.
For alternative techniques with greater detection sensitivity, perform silver
staining using the SilverXpress® Silver Staining Kit (see page 42) or SYBR®
Green I staining (see page 47).
48
Gel Drying
Introduction
Dry gels by passive evaporation (air-drying) or vacuum drying. Vacuum
drying is faster than passive air-drying methods but often results in cracked
gels due to the speed of dehydration.
We recommend drying Novex® Pre-Cast gels using passive air-drying methods
such as the DryEase® Mini-Gel Drying System (see below). For applications that
require vacuum drying, follow the recommendations on page 51 to minimize
cracking of the gels.
Materials Supplied
by the User
You will need the following items for drying your gel (see page 63 for ordering
information on Invitrogen products):
DryEase® Mini-Gel
Drying System

DryEase® Mini-Gel Drying System

Gel-Dry™ Drying Solution (or prepare your own gel drying solution
containing 30% methanol and 5% glycerol)

StainEase® Gel Staining Tray or a suitable round container
Silver stained and Coomassie stained Novex® Gels can be dried by vacuum
drying or by air-drying. We recommend using the DryEase® Mini-Gel Drying
System to air-dry the gel.
A brief gel drying protocol using the DryEase® Mini-Gel Drying System is
provided below. For more details on this system, refer to the DryEase® MiniGel Drying System manual (IM-2380). This manual is available for download
from our website at www.invitrogen.com or contact Technical Support (see
page 76).
1. After all staining and destaining steps are complete, wash the destained
gel(s) three times for two minutes each time in deionized water (50 mL per
mini-gel) on a rotary shaker.
2. Decant the water and add fresh Gel-Dry™ Drying Solution (35 mL per minigel).
3. Equilibrate the gel in the Gel-Dry™ Drying Solution by shaking the gel for
15–20 minutes in the StainEase® Gel Staining Tray or in a round container.
Note: Do not equilibrate gels stained with Coomassie G-250 in the GelDry™ Drying Solution for more than 5 minutes to avoid losing band
intensity.
4. Cut any rough edges off the gel (including the wells and the gel foot) using
the Gel Knife or a razor blade.
5. Remove 2 pieces (per gel) of cellophane from the package.
6. Immerse one sheet at a time in the Gel-Dry™ Drying Solution. Allow
10 seconds for complete wetting before adding additional sheets. Do not
soak the cellophane for more than 2 minutes.
Continued on next page
49
Gel Drying, Continued
DryEase® Mini-Gel
Drying System,
continued
7. Place one side of the DryEase® Gel Drying Frame with the corner pin facing
up, on the DryEase® Gel Drying Base.
8. Center a piece of pre-wetted cellophane from Step 5 over the base/frame
combination, so the cellophane lays over the inner edge of the frame.
9. Lay the gel on the center of the cellophane sheet making sure no bubbles are
trapped between the gel and the cellophane. Add some Gel-Dry™ Drying
Solution to the surface of the cellophane, if necessary.
10. Carefully lay the second sheet of cellophane over the gel so that no bubbles are
trapped between the cellophane and the gel. Add some Gel-Dry™ Drying
Solution if necessary. Gently smooth out any wrinkles in the assembly with a
gloved hand.
11. Align the remaining frame so that its corner pins fit into the appropriate holes
on the bottom frame. Push the plastic clamps onto the four edges of the
frames.
12. Lift the frame assembly from the DryEase® Gel Drying Base and pour off the
excess solution from the base.
13. Stand the gel dryer assembly upright on a bench top. Be careful to avoid drafts
as they can cause an uneven rate of dying which leads to cracking. Drying
takes between 12–36 hours depending on humidity and gel thickness.
14. When the cellophane is dry to touch, remove the gel/cellophane sandwich
from the drying frame. Trim off the excess cellophane.
15. Press the dried gel(s) between the pages of a notebook under light pressure for
approximately 2 days so they remain flat for scanning, photography, display,
and overhead projection.
Continued on next page
50
Gel Drying, Continued
Vacuum Drying
General guidelines are provided below to minimize cracking during vacuum
drying of gels. For detailed instructions on vacuum drying, follow the
manufacturer’s recommendations.
Handle Gels with Care:
Remove the gel from the cassette without breaking or tearing the edges. Small
nicks or tears can act as a starting point for cracking. Remove the gel wells and
foot off the bottom of the gel with a Gel Knife or a razor blade as described on
page 34. Use the StainEase® Staining Tray for staining and destaining gels. This
tray is designed to facilitate the solution changing process without handling of
gels.
Use a Gel Drying Solution:
We recommend equilibrating the gel in a gel drying solution such as Gel-Dry™
Gel Drying Solution for 10–30 minutes at room temperature with gentle
shaking on an orbital shaker before drying the gel. Gel-Dry™ Gel Drying
Solution contains a proprietary non-glycerol component to effectively regulate
the rate of drying and prevent cracking. The gel drying solutions do not
interfere with autoradiography.
To prepare your own gel drying solution, prepare a solution containing
30% methanol and 5% glycerol.
Note: Do not incubate gels stained with Coomassie G-250 in gel drying solution
for more than 5 minutes as the bands may fade.
Remove Air Bubbles:
Remove any air bubbles that may be trapped between the paper, gel, and
plastic wrap by rolling a small glass pipette over the gel. Use additional gel
drying solution to help remove the air bubbles.
Use Proper Gel Dryer Set-up:
Place gel on the gel dryer with the plastic wrap facing up. Make sure the
vacuum pump is in working condition, and properly set up to form a tight seal
when on. Use drying conditions for polyacrylamide gels, with the temperature
increasing to a set value and holding for the duration of the drying cycle. We
recommend drying mini-gels at 80C for 2 hours.
Ensure Gel is Completely Dry:
The gel will crack if the vacuum seal of the heated gel dryer is broken prior to
complete drying of the gel. To ensure the gel is completely dried before
releasing the vacuum seal, follow these tips :

Check the temperature of the gel
The temperature of the dried gel should be the same as the temperature of
the surrounding gel drying surface. If the temperature of the dried gel is
cooler, then the gel is not completely dried.

Check for moisture in the tubing connecting the gel dryer to the vacuum
pump
The gel is not completely dried if there is residual moisture in the tubing
and additional drying time is required.
51
Blotting Novex® Pre-Cast Gels
Introduction
After performing electrophoresis, proteins can be transferred to membranes for
subsequent analysis. Methods of transfer include wet, semi-wet, semi-dry, and
dry blotting. Semi-dry blotting can be performed with the Novex® Semi-Dry
Blotter, and dry blotting is performed with the iBlot® Gel Transfer Device. Refer
to the respective manuals for information on blotting with these devices.
Instructions are provided below for semi-wet blotting of Novex® Pre-Cast Gels
using the XCell II™ Blot Module. For more information on the XCell II™ Blot
Module, refer to the manual (IM-9051) available at www.invitrogen.com or
contact Technical Support (see page 76).
If you are using any other blotting apparatus, follow the manufacturer’s
recommendations.
Power
Considerations for
Blotting
During blotting, the distance traveled (gel thickness) between the electrodes is
much lower than during electrophoresis requiring lower voltage and lower
field strength (volts/distance). However, the cross sectional area of current
flow is much greater requiring higher current.
Blotting power requirements depend on field strength (electrode size) and
conductivity of transfer buffer. The higher the field strength and conductivity
of the buffer, the higher is the current requirement (the current decreases
during the run as the ions in the buffer polarize). It is important to use a power
supply capable of accommodating the initial high current requirement.
Materials Supplied
by the User
In addition to the XCell II™ Blot Module, the following reagents are needed for
blotting your gel (see page 63 for ordering information on Invitrogen products):
 Blotting membranes
 Filter paper (not needed if using Novex® pre-cut membrane/filter paper
sandwiches)
 Methanol (if using PVDF membranes)
 Appropriate Transfer Buffer
 Deionized water
Preparing Transfer
Buffer
For blotting Tris-Glycine, Tricine, and IEF Gels use 1X Tris-Glycine Transfer
Buffer. If you are preparing your own transfer buffer see page 66 for a recipe.
An alternate transfer protocol for IEF Gels is provided on page 57.
If you are performing protein sequencing, an alternate transfer buffer compatible
with the technique is listed on the next page.
Prepare 1,000 mL of Transfer Buffer:
Tris-Glycine Transfer Buffer (25X)
Methanol
Deionized Water
Total Volume
40 mL
200 mL
760 mL
1,000 mL
Continued on next page
52
Blotting Novex® Pre-Cast Gels, Continued
Preparing Transfer For blotting TBE and TBE-Urea Gels, use 0.5X TBE Running Buffer. If you are
Buffer for TBE Gels preparing your own transfer buffer, see page 69 for a recipe.
Prepare 1,000 mL of 1X Tris-Glycine Transfer Buffer using the Tris-Glycine
Transfer Buffer (25X) as follows:
TBE Running Buffer (5X)
Methanol
Deionized Water
Total Volume
40 mL
200 mL
760 mL
1,000 mL
For blotting TBE and TBE-Urea Gels
Dilute the 5X TBE Running Buffer to 0.5X with deionized water.
Preparing Transfer
Buffer Compatible
with Protein
Sequencing
Tris-Glycine Transfer Buffer interferes with protein sequencing. If you are
performing protein sequencing, use the NuPAGE® Transfer Buffer or the 0.5X
TBE Running Buffer to perform blotting.
The NuPAGE® Transfer Buffer protects against modification of the amino acid
side chains and is compatible with N-terminal protein sequencing using Edman
degradation.
Preparing Blotting
Pads
Use about 700 mL of 1X Transfer Buffer to soak the pads until saturated.
Remove the air bubbles by squeezing the pads while they are submerged in
buffer. Removing the air bubbles is essential as they can block the transfer of
biomolecules if they are not removed.
Preparing Transfer
Membrane and
Filter Paper
Cut the transfer membrane and filter paper to the dimensions of the gel or use
Novex® pre-cut membrane/filter paper sandwiches.

PVDF membrane—Pre-wet PVDF membrane for 30 seconds in methanol,
ethanol, or isopropanol. Briefly rinse in deionized water, then place in a
shallow dish with 50 mL of 1X Transfer Buffer for several minutes.

Nitrocellulose—Place the membrane directly into a shallow dish
containing 50 mL of 1X Transfer Buffer for several minutes.

Filter paper—Soak the filter paper briefly in 1X Transfer Buffer
immediately prior to use.

Gel—Use the gel immediately following the run. Do not soak the gel in
transfer buffer.
Continued on next page
53
Blotting Novex® Pre-Cast Gels, Continued
Western Transfer
Using the XCell II™
Blot Module
Wear gloves while performing the blotting procedure to prevent contamination
of gels and membranes, and exposure to irritants commonly used in
electrotransfer.
Transferring One Gel
1.
After opening the gel cassette as described on page 34, remove wells with
the Gel Knife.
2.
Place a piece of pre-soaked filter paper on top of the gel, with the edge
above the slot in the bottom of the cassette (leaving the foot of the gel
uncovered). Keep the filter paper saturated with the transfer buffer and
remove all trapped air bubbles by gently rolling over the surface using a
glass pipette as a roller.
3.
Turn the plate over so the gel and filter paper are facing downwards over a
gloved hand or clean flat surface.
4.
Use the Gel Knife to push the foot out of the slot in the plate, and separate
the gel from the plate.
5.
When the gel is on a flat surface, cut the foot off the gel with the Gel Knife.
6.
Wet the surface of the gel with transfer buffer and position the pre-soaked
transfer membrane on the gel, ensuring all air bubbles have been removed.
7.
Place another pre-soaked filter paper on top of the membrane. Remove any
trapped air bubbles.
8.
Place two soaked blotting pads into the cathode (–) core of the blot module.
The cathode core is the deeper of the two cores and the corresponding
electrode plate is a darker shade of gray. Carefully pick up the
gel/membrane assembly and place it on the pad such that the gel is closest
to the cathode plate (see Figure 1, next page).
9.
Add enough pre-soaked blotting pads to raise the assembly 0.5 cm over the
edge of cathode core. Place the anode (+) core on top of the pads. The
gel/membrane assembly should be held securely between the two halves
of the blot module ensuring complete contact of all components.
10. Position the gel membrane sandwich and blotting pads in the cathode core
of the XCell II™ Blot Module to fit horizontally across the bottom of the
unit. There should be a gap of approximately 1 cm at the top of the
electrodes when the pads and assembly are in place.
11. Hold the blot module together firmly and slide it into the guide rails on the
Lower Buffer Chamber. The blot module fits into the unit only one way,
with the (+) sign at the upper left hand corner of the blot module, and the
inverted gold post fitting into the connector on the right side of the Lower
Buffer Chamber.
12. Place the gel tension wedge so that its vertical face is against the blot
module. Lock the gel tension wedge by pulling the lever forward.
Continued on next page
54
Blotting Novex® Pre-Cast Gels, Continued
Western Transfer
Using the XCell II™
Blot Module,
continued
13. Fill the blot module with 1X Transfer Buffer until the gel/membrane
sandwich is covered in Transfer Buffer. To avoid generating extra
conductivity and heat, do not fill the chamber all the way to the top.
14. Fill the Lower Buffer Chamber with deionized water by pouring
approximately 650 mL in the gap between the front of the blot module and
the front of the Lower Buffer Chamber. The water level should reach
approximately 2 cm from the top of the Lower Buffer Chamber. This serves
to dissipate heat produced during the run.
15. Place the lid on top of the unit.
16. With the power turned off, plug the red and black leads into the power
supply. Refer to Recommended Transfer Conditions on the next page for
transfer conditions.
Transferring Two Gels in One Blot Module
+
1.
Repeat Steps 1–7 (previous page) twice to make two gel-membrane
assemblies.
2.
Place two pre-soaked pads on cathode shell of blot module. Place the first
gel/membrane assembly on the pads such that the gel faces the cathode
plate. (See Figure 2).
3.
Add another pre-soaked blotting pad on top of first gel/membrane
assembly.
4.
Position second gel/membrane assembly on top of blotting pad with the
gel facing the cathode side.
5.
Proceed with steps 8–13 from Transferring One Gel.
6.
Refer to Recommended Transfer Conditions on the next page for transfer
conditions.
Figure 1
Blotting Pad
Blotting Pad
+
Figure 2
Blotting Pad
Blotting Pad
Filter Paper
Filter Paper
Filter Paper
Transfer Membrane
Second Gel
Filter Paper
Blotting Pad
Blotting Pad
Blotting Pad
Filter Paper
Transfer Membrane
Gel
Transfer Membrane
First Gel
Filter Paper
Cathode Core (-)
Blotting Pad
Blotting Pad
Cathode Core (-)
Continued on next page
55
Blotting Novex® Pre-Cast Gels, Continued
Recommended
Transfer
Conditions
Gel
Tris-Glycine Gel
Tricine Gel
The transfer conditions for Novex® Pre-Cast Gels using the XCell II™ Blot
Module are listed in the table below.
Note: The expected current listed in the table is for transferring one gel. If you
are transferring two gels in the blot module, the expected current is roughly
twice the listed value.
Transfer Buffer
1X Tris-Glycine Transfer
Buffer with 20% methanol
Membrane
Nitrocellulose
or PVDF
Power Conditions
25 V constant for 1–2 hours
Expected Current
Start: 100 mA
IEF Gel
1X Tris-Glycine Transfer
Buffer with 20% methanol
Nitrocellulose
or PVDF
25 V constant for 1 hour
Expected Current
Start: 65–85 mA
0.7% Acetic acid pH 3.0
See next page for details on
this alternate transfer
protocol.
TBE Gel
0.5X TBE Running Buffer
Nitrocellulose
or PVDF
10 V constant for 1 hour
Expected Current
Start: 65–85 mA
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA
TBE-Urea Gel
0.5X TBE Running Buffer
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA
DNA Retardation
Gel
0.5X TBE Running Buffer
Nylon
30 V constant for 1 hour
Expected Current
Start: 39 mA
End: 35 mA
Continued on next page
56
Blotting Novex® Pre-Cast Gels, Continued
Blotting IEF Gels
Novex® IEF Gels are composed of 5% polyacrylamide and are more susceptible
to hydrolysis due to the heat generated with the recommended blotting
protocol. The following protocol has been optimized to prevent hydrolysis and
effective transfer of basic proteins due to the low pH of the transfer buffer.
1.
Prepare chilled 0.7% acetic acid.
2.
After electrophoresis, remove the gel from the cassette and equilibrate the
gel in the 0.7% acetic acid for 10 minutes.
Tip: The 5% polyacrylamide gels are stickier and more difficult to handle
than higher percentage polyacrylamide gels. To prevent the gel from
sticking to the filer paper before it is in the proper position, remove the gel
from the equilibration solution by submerging a piece of filter paper under
the gel while it is floating in the equilibration solution. When the gel and
filter paper are in the correct position, lift the filter paper so that it attaches
to the gel.
Blotting Native
Gels
3.
Assemble the gel/membrane sandwich as described on page 54, except in
reverse order so that the membrane is on the cathode (–) side of the gel.
4.
Transfer for 1 hour at 10 V constant.
During SDS-PAGE all proteins have a net negative charge due to the SDS in the
sample buffer and the running buffer. Proteins separated during native gel
electrophoresis do not have a net charge which may cause problems during the
transfer. Some native proteins may have a higher pI than the pH of the TrisGlycine Transfer Buffer used in standard transfer protocols. Guidelines are
provided below to increase the transfer efficiency of native proteins.

Increasing the pH of the transfer buffer to 9.2 (25 mM Tris Base, 25 mM
glycine, pH 9.2), allows proteins with pI below 9.2 to transfer towards the
anode electrode
 Place a membrane on both sides of the gel if you are using the regular TrisGlycine Transfer Buffer, pH 8.3. If there are any proteins that are more basic
than the pH of the transfer buffer, they will be captured on the membrane
placed on the cathode side of the gel
 Incubate the gel in 0.1% SDS for 15 minutes before blotting with Tris-Glycine
Transfer Buffer. The small amount of SDS will render enough charge to the
proteins so they can move unidirectionally towards the anode and in most
cases will not denature the protein
Native proteins may diffuse out of the membrane into the solution during the
blocking or antibody incubation steps, as the native proteins tend to be more
soluble. To prevent diffusion of the proteins out of the membrane, we
recommend fixing the proteins to the membrane by air drying the membrane or
incubating the membrane in 5–10% acetic acid for 15 minutes followed by rinsing
the membrane with deionized water and then air drying.
By performing any of these two fixing methods the proteins will be sufficiently
unfolded to expose hydrophobic sites and bind more efficiently to the membrane.
57
Calibrating Protein Molecular Weight
Introduction
The molecular weight of a protein can be determined based upon its relative
mobility by constructing a standard curve with protein standards of known
molecular weights.
The protein mobility in SDS-PAGE gels is dependent on the

Length of the protein in its fully denatured state,

SDS-PAGE buffer systems

Secondary structure of the protein
An identical molecular weight standard may have slightly different mobility
resulting in different apparent molecular weight when run in different SDSPAGE buffer systems.
If you are using the Novex® protein molecular weight standards, see the
apparent molecular weights of these standards on the Novex® Pre-Cast Gels
listed on the next page to determine an apparent molecular weight of your
protein.
Protein Secondary
Structure
When using SDS-PAGE for molecular weight determination, slight deviations
from the calculated molecular weight of a protein (calculated from the known
amino acid sequence) can occur due to the retention of varying degrees of
secondary structure in the protein, even in the presence of SDS. This
phenomenon is observed in highly organized secondary structures (such as
collagens, histones, or highly hydrophobic membrane proteins) and in
peptides, where the effect of local secondary structure and amino acid side
chains becomes magnified relative to the total size of the peptide.
Buffer Systems
Slight differences in protein mobilities also occur when the same proteins are
run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a
different pH, which affects the charge of a protein and its binding capacity for
SDS. The degree of change in protein mobility is usually small in natural
proteins but more pronounced with “atypical” or chemically modified proteins
such as pre-stained standards.
Continued on next page
58
Calibrating Protein Molecular Weight, Continued
Assigned Apparent
Molecular Weights
Values for apparent molecular weight of Novex® molecular weight standards are
derived from the construction of a calibration curve in the Tris-Glycine SDS-PAGE
System. We have now calculated and assigned apparent molecular weights for the
Novex® protein standards in several buffer systems. Remember to use the one that
matches your gel for the most accurate calibration of your protein.
The following charts summarize the approximate molecular weight values for the
Novex® protein molecular weight standards when run in different buffer systems.
You may generate calibration curves in your lab with any other manufacturer’s
standards.
Novex® Sharp Pre-stained Protein
Standard
Tris-Glycine Gels (4–20%)
Tricine Gels (10–20%)
Band 1
260 kDa
260 kDa
Band 2
160 kDa
160 kDa
Band 3
110 kDa
110 kDa
Band 4
80 kDa
80 kDa
Band 5
60 kDa
60 kDa
Band 6
50 kDa
50 kDa
Band 7
40 kDa
40 kDa
Band 8
30 kDa
30 kDa
Band 9
20 kDa
20 kDa
Band 10
15 kDa
15 kDa
Band 11
10 kDa
10 kDa
Band 12
Mark 12™ Unstained Standard
3.5 kDa
Tris-Glycine Gels (4–20%)
Tricine Gels (10–20%)
200 kDa
200 kDa
-Galactosidase
116.3 kDa
116.3 kDa
Phosphorylase B
97.4 kDa
97.4 kDa
Bovine Serum Albumin
66.3 kDa
66.3 kDa
Glutamic Dehydrogenase
55.4 kDa
55.4 kDa
Lactate Dehydrogenase
36.5 kDa
36.5 kDa
31 kDa
31 kDa
Trypsin Inhibitor
21.5 kDa
21.5 kDa
Lysozyme
14.4 kDa
14.4 kDa
Aprotinin
6 kDa
6 kDa
Unresolved Insulin
3.5 kDa
Myosin
Carbonic Anhydrase
Insulin B Chain
Insulin A Chain
2.5 kDa
Continued on next page
59
Calibrating Protein Molecular Weight, Continued
Assigned Apparent Molecular Weights, continued
SeeBlue® Pre-Stained Standard
Tris-Glycine Gel (4–20%)
Tricine Gel (10–20%)
Myosin
250 kDa
210 kDa
BSA
98 kDa
78 kDa
Glutamic Dehydrogenase
64 kDa
55 kDa
Alcohol Dehydrogenase
50 kDa
45 kDa
Carbonic Anhydrase
36 kDa
34 kDa
Myoglobin
30 kDa
23 kDa
Lysozyme
16 kDa
16 kDa
Aprotinin
6 kDa
7 kDa
Insulin
4 kDa
4 kDa
SeeBlue® Plus2 Pre-Stained Standard
60
Tris-Glycine Gel (4–20%)
Tricine Gel (10–20%)
Myosin
250 kDa
210 kDa
Phosphorylase B
148 kDa
105 kDa
BSA
98 kDa
78 kDa
Glutamic Dehydrogenase
64 kDa
55 kDa
Alcohol Dehydrogenase
50 kDa
45 kDa
Carbonic Anhydrase
36 kDa
34 kDa
Myoglobin
22 kDa
17 kDa
Lysozyme
16 kDa
16 kDa
Aprotinin
6 kDa
7 kDa
Insulin
4 kDa
4 kDa
Troubleshooting
Introduction
Observation
Review the information below to troubleshoot your experiments with Novex®
Gels.
Cause
Solution
Run taking longer
time
Running buffer too dilute
Make fresh running buffer as described in this
manual and avoid adjusting the pH of the 1X
running buffer.
Low or no current
during the run
Incomplete circuit
 Remove the tape from the bottom of the
cassette prior to electrophoresis.
 Make sure the buffer covers the sample
wells.
 Check the wire connections on the buffer
core to make sure the connections are intact.
Faint shadow or
“ghost” band below
the expected protein
band
Ghost bands are caused due to
a slight lifting of the gel from
the cassette resulting in
trickling of some sample
beyond its normal migration
point. Gel lifting off the
cassette is caused due to:
 Expired gels
 Improper storage of gels
Streaking of proteins
Bands in the outer
lane of the gel are
curving upwards
 Avoid using expired gels. Use fresh gels
 Store the gels at the appropriate temperature
(see page v).
 Sample overload
 Load the appropriate amount of protein as
described on page 8.
 High salt concentration in
the sample
 Decrease the salt concentration of your
sample using dialysis or gel filtration
 Sample precipitates
 Increase the concentration of SDS in your
sample if necessary, to maintain the
solubility of the protein.
 Contaminants such as
membranes or DNA
complexes in the sample
 Centrifuge or clarify your sample to remove
particulate contaminants
 Concentrated buffer used
 The pre-made buffers are supplied as
concentrate. Dilute the buffers as described
in this manual.
 Expired gels used
 Avoid using gels after the expiration date.
 High voltage used
 Electrophorese the gel using conditions
described on page 33.
Continued on next page
61
Troubleshooting, Continued
Observation
Cause
Solution
Bands in the outside lanes
of the gel “smiling”
Expired gels used causing the
acrylamide to break down in the gel
Avoid using gels after the
expiration date. Use fresh gels.
Bands are running as U
shape rather than a flat
band
Samples are loaded on the gel and
not electrophoresed immediately
resulting in sample diffusion
Load samples on to the gel
immediately before electrophoresis.
Bands appear to be
“funneling” or getting
narrower as they progress
down the gel
Proteins are over-reduced causing
the proteins to be negatively
charged and repel each other.
Reduce the proteins using DTT or
-mercaptoethanol as described on
page 10.
Dumbbell shaped bands
after electrophoresis
Loading a large volume of sample
causing incomplete stacking of the
entire sample. This effect is
intensified for larger proteins
Load the appropriate volume of
sample per well as described on
page 8. If your sample is too dilute,
concentrate the sample using salt
precipitation or ultrafiltration.
For TBE-Urea gels
 RNase contamination
High background and
smeared bands or
abnormal band shapes
 Always wear gloves and use
sterile techniques to prevent
RNase contamination.
 Sample renatured
 Heat the sample for 3 minutes at
70C and keep the sample in ice
to prevent renaturation. Proceed
to electrophoresis immediately
after loading.
 Sample overloaded
 Recommended DNA load is
0.16–0.33 μg/band.
 Urea not completely flushed from
the wells
 Be sure to thoroughly flush urea
out of the wells prior to loading
the sample.
62
Appendix
Accessory Products
Electrophoresis
Reagents
Ordering information on a variety of electrophoresis reagents and apparatus
available from Invitrogen is provided below. For more information, visit our
website at www.invitrogen.com or call Technical Support (see page 76).
Product
Quantity
™
XCell SureLock Mini-Cell
™
XCell II Blot Module
®
PowerEase 500 Power Supply
®
DryEase Mini-Gel Drying System
®
StainEase Staining Tray
™
Gel-Dry Drying Solution
®
iBlot Gel Transfer Device
Catalog no.
1 unit
EI0001
1 unit
EI9051
1 unit
EI8600
1 kit
NI2387
2/pack
NI2400
500 mL
LC4025
1 unit
IB1001
®
1 unit
SD1000
®
Novex Tris-Glycine SDS Running Buffer (10X)
500 mL
LC2675
NuPAGE® Sample Reducing Agent (10X)
250 μL
NP0004
NuPAGE® LDS Sample Buffer (4X)
250 mL
NP0008
Novex® Tris-Glycine Transfer Buffer (25X)
Novex Semi-Dry Blotter
500 mL
LC3675
®
500 mL
LC2672
®
20 mL
LC2673
®
20 mL
LC2676
®
500 mL
LC1675
®
20 mL
LC1676
®
500 mL
LC2670
®
500 mL
LC2671
®
1L
LC6675
®
10 mL
LC6678
®
10 mL
LC6876
20 mL
LC6877
NuPAGE Novex 4-12% Bis-Tris ZOOM Gel
1 gel
NP0330BOX
Novex® 4-20% Tris-Glycine ZOOM® Gel
1 gel
EC60261BOX
Novex® pH 3-7 IEF Buffer Kit (includes LC5300, LC5370, LC5371)
1 kit
LC5377
Novex® pH 3-10 IEF Buffer Kit (includes LC5300, LC5310, LC5311)
1 kit
LC5317
UltraPure Agarose
100 g
15510-019
Nitrocellulose (0.45μm)
20 membrane/filter papers
LC2000
Invitrolon PVDF (0.45 μm)
20 membrane/filter papers
LC2005
Nylon (0.45 μm)
20 membrane/filter papers
LC2003
Novex Tris-Glycine Native Running Buffer (10X)
Novex Tris-Glycine Native Sample Buffer (2X)
Novex Tris-Glycine SDS Sample Buffer (2X)
Novex Tricine SDS Running Buffer (10X)
Novex Tricine SDS Sample Buffer (2X)
Novex Zymogram Renaturing Buffer (10X)
Novex Zymogram Developing Buffer (10X)
Novex TBE Running Buffer (5X)
Novex Hi-Density TBE Sample Buffer (5X)
Novex TBE-Urea Sample Buffer (2X)
®
Novex Prep TBE-Urea Sample Buffer (2X)
®
®
™
™
®
Continued on next page
63
Accessory Products, Continued
Protein Stains and
Standards
Ordering information for stains and protein molecular weight standards is
provided below. For more information, visit our website at
www.invitrogen.com or contact Technical Support (see page 76).
Product
Application
Quantity
Catalog no.
SimplyBlue™ Safe-Stain
Fast, sensitive, safe Coomassie G-250
staining of proteins in polyacrylamide gels
1L
LC6060
SilverQuest™ Silver Staining
Kit
Sensitive silver staining of proteins
compatible with mass spectrometry
analysis
1 Kit
LC6070
Colloidal Blue Staining Kit
Sensitive colloidal Coomassie G-250
staining of proteins in polyacrylamide gels
1 Kit
LC6025
SilverXpress® Silver
Staining Kit
High-sensitivity, low background protein
and nucleic acid silver staining
1 Kit
LC6100
Mark 12™ Unstained
Standard
For estimating the apparent molecular
weight of proteins
1 mL
LC5677
MagicMark™ Western
Standard
For protein molecular weight estimation on
western blots
250 μL
LC5600
SeeBlue® Pre-Stained
Standard
For monitoring the progress of your run
and evaluating transfer efficiency
500 μL
LC5625
SeeBlue® Plus2 Pre-Stained
Standard
For visualizing protein molecular weight
range and evaluating transfer efficiency
500 μL
LC5925
Novex® Sharp Pre-stained
Protein Standard
For visualizing protein molecular weight
range and evaluating transfer efficiency
2 × 250 μL
LC5800
BenchMark™ Protein
Ladder
For estimating the apparent molecular
weight of proteins
2 × 250 μL
10747-012
IEF Marker 3-10
For determining the pI of proteins
500 μL
39212-01
Nucleic Acid
Markers
64
A large variety of nucleic acid markers are available from Invitrogen. ReadyLoad™ format (pre-mixed with loading buffer) nucleic acid markers are also
available for your convenience. For more information, visit our website at
www.invitrogen.com or contact Technical Support (see page 76).
Recipes
Tris-Glycine SDS
Running Buffer
The Tris-Glycine SDS Running Buffer is available from Invitrogen (see page 63).
25 mM Tris Base
192 mM Glycine
0.1% SDS
pH 8.3
1.
To prepare 1,000 mL of 10X Tris-Glycine SDS Running Buffer, dissolve the
following reagents to 900 mL ultrapure water:
Tris Base
29 g
Glycine
144 g
SDS
Tris-Glycine Native
Running Buffer
10 g
2.
Mix well and adjust the volume to 1,000 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For electrophoresis, dilute this buffer to 1X with water (see page 12). The pH of
the 1X solution is 8.3. Do not use acid or base to adjust the pH.
The Tris-Glycine Native Running Buffer is available from Invitrogen (see page 63).
25 mM Tris base
192 mM Glycine
pH 8.3
1.
To prepare 1,000 mL of 10X Tris-Glycine Native Running Buffer, dissolve the
following reagents to 900 mL ultrapure water:
Tris Base
29 g
Glycine
144 g
2.
Mix well and adjust the volume to 1,000 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For native electrophoresis, dilute this buffer to 1X with water (see page 12). The
pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.
Continued on next page
65
Recipes, Continued
Tris-Glycine SDS
Sample Buffer
The Tris-Glycine SDS Sample Buffer is available from Invitrogen (see page 63).
63 mM Tris HCl
10% Glycerol
2% SDS
0.0025% Bromophenol Blue
pH 6.8
1.
To prepare 10 mL of 2X Tris-Glycine SDS Sample Buffer, mix the following
reagents :
0.5 M Tris-HCl, pH 6.8
Glycerol
10% (w/v) SDS
0.1% (w/v) Bromophenol Blue
Tris-Glycine Native
Sample Buffer
2.
Adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The Tris-Glycine Native Sample Buffer is available from Invitrogen (see page 63).
1X composition
100 mM Tris HCl
10% Glycerol
0.0025% Bromophenol Blue
pH 8.6
1.
To prepare 10 mL of 2X Tris-Glycine Native Sample Buffer, mix the following
reagents :
0.5 M Tris HCl, pH 8.6
Glycerol
0.1% (w/v) Bromophenol Blue
Tris-Glycine
Transfer Buffer
2.5 mL
2 mL
4 mL
0.5 mL
4 mL
2 mL
0.5 mL
2.
Adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The Tris-Glycine Transfer Buffer is available from Invitrogen (see page 63).
12 mM Tris Base
96 mM Glycine
pH 8.3
1.
To prepare 500 mL of 25 × Tris-Glycine Transfer Buffer, dissolve the
following reagents in 400 mL ultrapure water:
Tris Base
Glycine
18.2 g
90 g
2.
Mix well and adjust the volume to 500 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For blotting, dilute this buffer as described on page 52. The pH of the 1X
solution is 8.3. Do not use acid or base to adjust the pH.
Continued on next page
66
Recipes, Continued
Tricine SDS
Sample Buffer
The Tricine SDS Sample Buffer is available from Invitrogen (see page 63).
450 mM Tris HCl
12% Glycerol
4% SDS
0.0025% Coomassie Blue G
0.0025% Phenol Red
pH 8.45
1.
To prepare 10 mL of 2X Tricine SDS Sample Buffer, mix the following reagents:
3 M Tris HCl, pH 8.45
Glycerol
SDS
Tricine SDS
Running Buffer
2.4 mL
0.8 g
0.1% Coomassie Blue G
0.5 mL
0.1% Phenol Red
0.5 mL
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The Tricine SDS Running Buffer is available from Invitrogen (see page 63).
100 mM Tris base
100 mM Tricine
0.1% SDS
pH 8.3
1.
To prepare 1,000 mL of 10 × Tricine SDS Running Buffer, dissolve the following
reagents in 900 mL deionized water:
Tris Base
121 g
Tricine
179 g
SDS
10X Zymogram
Renaturing Buffer
3 mL
10 g
2.
Mix well and adjust the volume to 1,000 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of
the 1X solution is 8.3. Do not use acid or base to adjust the pH.
The Zymogram Renaturing Buffer is available from Invitrogen (see page 63).
25% (v/v) Triton® X-100
1.
To prepare 500 mL of 10X Zymogram Renaturing Buffer, add 125 mL
Triton® X-100 to 300 mL ultra pure water.
2.
Mix well and adjust the volume to 500 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
Continued on next page
67
Recipes, Continued
Zymogram
Developing Buffer
The Zymogram Developing Buffer is available from Invitrogen (see page 63).
50 mM Tris base
40 mM HCl
200 mM NaCl
5 mM CaCl2
0.02% (w/v) Brij 35
1.
IEF Sample Buffer
pH 3–7
Tris Base
30.2 g
6N HCl
33 mL
NaCl
58.5 g
CaCl2.2H2O
3.7 g
Brij 35
1.0 g
2.
Mix well and adjust the volume to 500 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For developing the zymogram gel, dilute this buffer to 1X with water (see
page 18).
The IEF Sample Buffer pH 3–7 is available from Invitrogen (see page 63).
40 mM Lysine (free base)
15% Glycerol
1.
IEF Sample Buffer,
pH 3–10
To prepare 500 mL of 10X Zymogram Developing Buffer, dissolve the
following reagents in 400 mL deionized water:
To prepare 10 mL of 2X IEF Sample Buffer pH 3–7, mix the following
reagents:
10X IEF Cathode Buffer, pH 3–7 (see next page)
2 mL
Glycerol
3 mL
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The IEF Sample Buffer pH 3–10 is available from Invitrogen (see page 63).
20 mM Lysine (free base)
20 mM Arginine (free base)
15% Glycerol
1.
To prepare 10 mL of 2X IEF Sample Buffer pH 3–10, mix the following
reagents:
10X IEF Cathode Buffer, pH 3–10 (see next page)
2 mL
Glycerol
3 mL
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
Continued on next page
68
Recipes, Continued
IEF Cathode Buffer, The IEF Cathode Buffer pH 3–7 is available from Invitrogen (see page 63).
pH 3–7
40 mM Lysine (free base)
1.
To prepare 100 mL of 10X IEF Cathode Buffer pH 3–7, dissolve 5.8 g of
Lysine (free base) in 100 mL of ultrapure water.
2.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
IEF Cathode Buffer, The IEF Cathode Buffer pH 3–10 is available from Invitrogen (see page 63).
pH 3–10
20 mM Lysine (free base)
20 mM Arginine (free base) You can use D, L, or D/L form of arginine
pH 10.1
IEF Anode Buffer
1.
To prepare 100 mL of 10X IEF Cathode Buffer pH 3–10, dissolve 2.9 g of
Lysine (free base) and 3.5 g of Arginine (free base) in 100 mL of ultrapure
water.
2.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The IEF Anode Buffer is available from Invitrogen (see page 63).
7 mM Phosphoric acid
TBE Running
Buffer
1.
To prepare 100 mL of 50X IEF Anode Buffer, mix 2.4 mL of 85% phosphoric
acid with 97.6 mL of ultrapure water.
2.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
The TBE Running Buffer is available from Invitrogen (see page 63).
89 mM Tris base
89 mM Boric acid
2 mM EDTA (free acid)
pH 8.3
1.
To prepare 1,000 mL of 5X TBE Running Buffer, dissolve the following
reagents in 900 mL deionized water:
Tris Base
Boric acid
EDTA (free acid)
54 g
27.5 g
2.9 g
2.
Mix well and adjust the volume to 1,000 mL with ultrapure water.
3.
Store at room temperature. The buffer is stable for 6 months when stored at
room temperature.
4.
For electrophoresis, dilute this buffer to 1X with water as described on
page 26. The pH of the 1X solution is 8.3. Do not use acid or base to adjust
the pH.
Continued on next page
69
Recipes, Continued
Hi-Density TBE
Sample Buffer
The Hi-Density TBE Sample Buffer is available from Invitrogen (see page 63).
18 mM Tris base
18 mM Boric acid
0.4 mM EDTA (free acid)
3% Ficoll® Type 400
0.02% Bromophenol Blue
0.02% Xylene Cyanol
1.
To prepare 10 mL of 5X Hi-Density TBE Sample Buffer, dissolve the
following reagents in 9 mL deionized water:
5X TBE Running Buffer (see previous page)
®
TBE-Urea Sample
Buffer
2 mL
Ficoll Type 400
1.5 g
1% Bromophenol Blue
1 mL
1% Xylene Cyanol
1 mL
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
The TBE-Urea Sample Buffer is available from Invitrogen (see page 63).
45 mM Tris base
45 mM Boric acid
1 mM EDTA (free acid)
6% Ficoll® Type 400
3.5 M Urea
0.005% Bromophenol Blue
0.025% Xylene Cyanol
1.
To prepare 10 mL of 2X TBE-Urea Sample Buffer, dissolve the following
reagents in 9 mL deionized water:
5X TBE Running Buffer (see previous page)
®
2 mL
Ficoll Type 400
1.2 g
1% Bromophenol Blue
1 mL
1% Xylene Cyanol
Urea
0.5 mL
4.2 g
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 3 months when stored at +4C.
Continued on next page
70
Recipes, Continued
Prep TBE–Urea
Sample Buffer
The Prep TBE–Urea Sample Buffer is available from Invitrogen (see page 63).
45 mM Tris base
45 mM Boric acid
1 mM EDTA (free acid)
6% Ficoll® Type 400
3.5 M Urea
1.
To prepare 10 mL of 2X Prep TBE–Urea Sample Buffer, dissolve the
following reagents in 9 mL deionized water:
5X TBE Running Buffer (see page 69)
®
2 mL
Ficoll Type 400
1.2 g
Urea
4.2 g
2.
Mix well and adjust the volume to 10 mL with ultrapure water.
3.
Store at +4C. The buffer is stable for 6 months when stored at +4C.
71
Gel Migration Charts
Novex® TrisGlycine Gel
Migration Chart
The migration patterns of protein standards* on Novex® Tris-Glycine Gels are
shown on the table below. Use the table to select the proper gel for separating
proteins based on size. Optimal resolution is achieved when protein bands migrate
within the shaded regions.
TTris-Glycine
Tris-Gl
ycine Gels
Large Proteins
(116-500 kDa)
4%
6%
Mid-Size Proteins
(20-250 kDa)
8%
10%
12%
Small Proteins
(3-60 kDa)
14%
16%
Wide Range
(6-200 kDa)
18%
4-12%
8-16%
4-20%
10
10-20%
200 kDa
200 kDa
20
200 kDa
200 kDa
200 kDa
116 kDa
30
97 kDa
116 kDa
200 kDa
200 kDa
200 kDa
116 kDa
116 kDa
97 kDa
97 kDa
66 kDa
55 kDa
66 kDa
% of length of gel
66 kDa
55 kDa
200 kDa
36 kDa
116 kDa
55 kDa
97 kDa
31 kDa
36 kDa
200 kDa
66 kDa
55 kDa
36 kDa
66 kDa
50
200 kDa
55 kDa
97 kDa
116 kDa
97 kDa
97 kDa
66 kDa
66 kDa
40
116 kDa
116 kDa
116 kDa
31 kDa
97 kDa
55 kDa
36 kDa
97 kDa
21 kDa
116 kDa
66 kDa
200 kDa
31 kDa
55 kDa
60
36 kDa
116 kDa
31 kDa
97 kDa
21 kDa
14 kDa
36 kDa
55 kDa
31 kDa
66 kDa
21 kDa
70
31 kDa
21 kDa
66 kDa
14 kDa
21 kDa
6 kDa
97 kDa
55 kDa
36 kDa
14 kDa
55 kDa
31 kDa
36 kDa
80
14 kDa
6 kDa
36 kDa
3.5 kDa
21 kDa
14 kDa
6 kDa
21 kDa
6 kDa
90
31 kDa
31 kDa
66 kDa
3.5 kDa
116 kDa
2.5 kDa
14 kDa
6 kDa
100
55 kDa
14 kDa
6 kDa
21 kDa
36 kDa
* Bands correspond to the migration of Mark12™ Unstained Standard under denaturing conditions.
Continued on next page
72
Gel Migration Charts, Continued
The migration patterns of protein markers on Novex® Tricine, IEF, and
Zymogram Gels are shown on the table below. Optimal resolution is achieved
when protein bands migrate within the shaded regions.
T
Tricine
Gels (Peptides)*
Blotting &
Sequencing
10%
Synthetic
Peptides
& Tryptic
Analysis
16%
Wide
Range
10-20%
IEF Gels (pI)
Zymogram Gels (Proteases)**
Isoelectric Point
& 2D Analysis
pH
3-10
pH
3-7
Protease Analysis
10% Gel
(w/gelatin)
12% Gel
(w/casein)
4-16% Gel
(w/pre-stained
casein blue)
10
200 kDa
116 kDa
200 kDa
20
116 kDa
30
97 kDa
97 kDa
pI 7.80
66 kDa
55 kDa
200 kDa
36 kDa
116 kDa
pI 6.50
97 kDa
31 kDa
66 kDa
1
66 kDa
pI 6.00
40
55 kDa
21 kDa
55 kDa
14 kDa
% of length of gel
Novex® Tricine, IEF,
and Zymogram Gel
Migration Chart
50
36 kDa
pI 5.10
pI 6.50
31 kDa
2 1
36 kDa
60
31 kDa
6 kDa
21 kDa
14 kDa
pI 4.65
3 3
3.5 kDa
70
21 kDa
2.5 kDa
4
3
6 kDa
80
2
2
pI 6.00
4
2
14 kDa
3.5 kDa
2.5 kDa
pI 4.65
4
90
6 kDa
pI 3.50
4
100
* Bands correspond to the migration of Mark12™ Unstained Standard under denaturing conditions.
** The numbered bands on the Zymogram Gel patterns refer to the following proteases: Band 1: Collagenase
Type I (140 kDa); Band 2: Thermolysin (37 kDa); Band 3: Chymotrypsin (30 kDa); Band 4: Trypsin (19 kDa)
Continued on next page
73
Gel Migration Charts, Continued
Novex® TBE and
TBE-Urea Gel
Migration Chart
The migration patterns of DNA fragments on Novex® TBE and TBE-Urea Gels are
shown on the table below. Optimal resolution is achieved when nucleic acid bands
migrate within the shaded regions.
T B E G el s
Oligonucleotides
Restriction Digest
PCR Products
6%
8%
10%
20%
T B E -U r ea G el s
Restriction Digest
PCR Products
DNA
Retardation
4-12%
6%
4-20%
2645 bp
2645 bp
1605 bp
20
1605 bp
1198 bp
1198 bp
30
1605 bp
40
% of length of gel
179 bp
350 bp
126 bp
676 bp
517 bp
126 bp
80
100
350 bp
80
42
350 bp
75 bp
58
222 bp
179 bp
100
222 bp
350 bp
179 bp
126 bp
30
126 bp
36 bp
126 bp
58
160
517 bp
51 bp
65 bp
200
460 bp
75 bp
70
100
676 bp
676 bp
179 bp
65 bp
126 bp
179 bp
1198 bp
517 bp
460 bp
60
200
460 bp
222 bp
179 bp
300
1605 bp
460 bp
222 bp
350 bp
50
400
1198 bp
350 bp
222 bp
222 bp
1605 bp
350 bp
460 bp
1605 bp
300
1198 bp
517 bp
517 bp
460 bp
2645 bp
2645 bp
500
200
517 bp
460 bp
15%
300
500
676 bp
676 bp
517 bp
10%
2645 bp
2645 bp
676 bp
676 bp
6%
Oligo
Size
Check
780
1198 bp
1605 bp
Large
Oligos
500
2645 bp
10
RNase
Protection
Assays
42
80
222 bp
75 bp
179 bp
51 bp
75 bp
65 bp
51 bp
21 bp
80
36 bp
18 bp
75 bp
90
51 bp
30
21
65 bp
75 bp
51 bp
65 bp
36 bp
21 bp
42
21
18 bp
51 bp
36 bp
100
58
36 bp
65 bp
51 bp
75 bp
126 bp
30
36 bp
Values are given in base pairs (bp)
Values are given in bases
Continued on next page
74
Gel Migration Charts, Continued
The migration patterns of protein standards* on ZOOM® Gels are shown on the
table below. Optimal resolution is achieved when protein bands migrate within
the shaded regions.
Zoom® Gels
4-12%
4-12%
Bis-Tris
Bis-Tris
Zoom® Gel Zoom® Gel
w/ MES
w/ MOPS
Running
Running
Buffer
Buffer
4-20%
Tris-Glycine
Zoom® Gel
10
200 kDa
200 kDa
20
116 kDa
200 kDa
97 kDa
30
66 kDa
116 kDa
97 kDa
116 kDa
55 kDa
97 kDa
40
% of length of gel
ZOOM® Gel
Migration Chart
66 kDa
66 kDa
55 kDa
50
36 kDa
31 kDa
55 kDa
36 kDa
60
31 kDa
21 kDa
70
14 kDa
36 kDa
21 kDa
31 kDa
14 kDa
80
6 kDa
6 kDa
21 kDa
90
3.5 kDa
14 kDa
2.5 kDa
100
* On ZOOM® Gels, migration of bands
correspond to the migration of Mark12™
Unstained Standard (Cat. no. LC5677)
under denaturing conditions.
75
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Technical Support, Continued
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References
Kubo, K. (1995). Effect of Incubation of Solutions of Proteins Containing Dodecyl Sulfate on the Cleavage
of Peptide Bonds by Boiling. Anal. Biochem. 225, 351-353.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage
T4. Nature 227, 680-685.
Ornstein, L. (1964). Disc Electrophoresis, 1, Background and Theory. Ann New York Acad. Sci 121, 321349.
Revzin, A. (1989). Gel Electrophoresis Assays for DNA-Protein Interactions. BioTechniques 4, 346-355.
Schaegger, H., and von Jagow, G. (1987). Tricine-Sodium dodecyl sulfate-Polyacrylamide Gel
Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal. Biochem. 166,
368-379.
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property of Life Technologies Corporation or their respective owners.
Ficoll® is a registered trademark of GE Healthcare.
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